Monthly Archives: October 2012

Methods To Differentiate Adipose Derived Stem Cells Into The Chondrogenic Phenotype

Me: This post will be critical and super important because it really does show the height increase seeker just which types of growth hormone combinations and mixtures will lead to the best and most optimum levels of chondrogenesis. Once of the biggest challenges and questions I had in my past self derived methods on height increase was what the growth factor combination should be. I had originally thought of the idea of injecting implants of slow releasing capsule stilled with growth factors in a series, similar to how the cocktail formula vaccine approach was done for HIV. However, I was not sure which BMPs and GDFs should go where. This will definitely push the effort and endeavor a big step forward.

It also contains actual step by step instruction on how to isolate and extract adipose derived stem cells from a subject and how to use basic chemistry lab techniques to form the chondrocytes. This may be a neccesary step in being able to test and apply any proposed ideas.

What this post does is to show what would be the actual reagents and procedure steps needed to differentiate adipose derived stem cells into the chondrogenic phenotype and then cartilage. I would guess the amount of equipment and materials needed would run up to $5-10K and the entire procedure would take 10-12 weeks. Of course at the very end you must do histological, immunohistological, and immunochemical testing to see how well was the overall yield.

From the full text of PubMed study link HERE…

Nat Protoc. Author manuscript; available in PMC 2011 November 17.

Published in final edited form as:

Nat Protoc. 2010 July; 5(7): 1294–1311.

Published online 2010 June 17. doi: 10.1038/nprot.2010.81

PMCID: PMC3219531

NIHMSID: NIHMS312303

Isolation of adipose derived stem cells and their induction to a chondrogenic phenotype

Bradley T. Estes,^† Brian O. Diekman,^† Jeffrey M. Gimble,^** and Farshid Guilak^†^*

Author information ► Copyright and License information ►

The publisher’s final edited version of this article is available at Nat Protoc

See other articles in PMC that cite the published article.

Summary

The ability to isolate, expand, and differentiate adult stem cells into a chondrogenic lineage is an important step in the development of tissue engineering approaches for cartilage repair or regeneration for the treatment of joint injury or osteoarthritis, or for application in plastic or reconstructive surgery. Adipose-derived stem cells (ASCs) provide an abundant and easily accessible source of adult stem cells for use in such regenerative approaches. This protocol describes the isolation of ASCs from liposuction aspirate, as well as cell culture conditions for growth factor based induction of ASCs into chondrocyte-like cells. These methods are similar to those used for bone marrow mesenchymal stem cells but distinct due to the unique properties of ASCs. Investigators can expect consistent ASC differentiation, allowing for slight variation due to donor and serum lot effects. Approximately 10–12 weeks are needed for ASC isolation and the characterization of chondrocyte-like cells, which is also described.

INTRODUCTION

The treatment of pathologies in articular and elastic cartilage pose important unmet challenges to the medical community. For example, arthritis represents the most common cause of disability in the US, leading to joint pain and dysfunction in over 40 million Americans ^1, 2 Osteoarthritis, the most common form of arthritis, involves degeneration of the articular cartilage, the smooth, load-bearing tissue lining the ends of long bones within the synovial joints of the body. The greatest risk factors for osteoarthritis include aging, obesity, joint trauma, and mutations in cartilage specific matrix proteins ³. Current estimates on the treatment costs, both indirect and direct, of osteoarthritis in the US are escalating to greater than $65 billion annually ⁴. For plastic and reconstructive surgery in the head and neck area, elastic cartilage is often needed for nose, ear, and trachea reconstruction ⁵. Estimates in the number of procedures involving bone and cartilage replacement exceed one million procedures per year ⁶.

Cartilage Properties and Current Treatment Options

The unique function and properties of cartilage are provided by the tissue’s extracellular matrix, which is maintained by a population of cells known as chondrocytes. Due to the small volume of chondrocytes (2–5% by volume), as well as the avascular and aneural properties of the tissue, cartilage exhibits little to no intrinsic repair capabilities in response to injury or disease. Traditional efforts to treat cartilage damage include joint lavage, tissue debridement, microfracture of the subchondral bone, abrasion arthroplasty, or the transplantation of autologous or allogeneic osteochondral grafts ^7–17. While these procedures have yielded promising clinical results, they are generally not applicable for large cartilage defects or for degenerative joint diseases such as osteoarthritis ^18–20. One tissue engineering approach, autologous chondrocyte transplantation, has shown promising results in early clinical reports, ^21, 22 but recent randomized, controlled trials suggest little difference in the efficacy of this procedure over microfracture of the subchondral bone ²³. In this regard, there has been significant interest in the development of new tissue engineering strategies for the repair or replacement of damaged or diseased cartilage, and integral to such approaches is the need for an abundant and easily accessible source of cells. While most early attempts at cartilage tissue engineering have relied on the use of primary differentiated chondrocytes, it is important to note that there are many issues associated with the harvest of autologous tissue and cells for repair of cartilage. Among these are the disease state of the harvested cells (from the inflamed joint), the potential for initiation of osteoarthritic changes in the joint ^23, 24, the lack of adequate autogenous tissue, and difficulty in expanding the cells ex vivo while maintaining the chondrocyte phenotype ^25, 26. These issues provide significant barriers for the use of autogenous primary chondrocytes for successful cartilage repair treatment strategies. Currently, defects in the head and neck area are typically treated with autologous cartilage, usually harvested from costal cartilage grafts ²⁷. Advantageously, the use of autologous tissues presents an implant that can be formed in the correct anatomical shape without risk of immunological rejection. However, the limitations and potential disadvantages of autologous tissue use are noteworthy and include lack of available tissue for large defects, the risk of iatrogenic deformity, and significant donor-site morbidity ^27, 28.

Stem Cells in Cartilage Repair

The use of adult stem cells presents a viable alternative for cartilage repair strategies and has the potential for success while avoiding the issues associated with autogenous grafts andor cells. Mesenchymal stem cells (MSCs) from bone marrow have been shown to possesses multilineage differentiation potential and have been characterized extensively for a variety of tissue engineering applications including chondrogenesis ^29–31. Another multipotent adult stem cell population, adipose derived stem cells (ASCs), can easily be obtained from liposuction waste ^32, 33 and has been shown in numerous studies to exhibit the potential for chondrogenesis, osteogenesis, adipogenesis, myogenesis, and some aspects of neurogenesis ^34–45. While these cells show some similarities to bone marrow MSCs, they appear to have a number of distinct characteristics with respect to their cell surface markers, differentiation potential, and abundance in the body. For example, compared to 100 ml of bone marrow aspirate, up to 300-fold more stem cells can be obtained from 100 g of adipose tissue ^31, 46.

ASCs have demonstrated significant potential for chondrogenic differentiation when expanded in appropriate monolayer conditions ⁴⁷ and cultured in growth factor containing medium in 3D culture^{34–36, 43, 45}. As it was originally hypothesized that ASCs were another source of MSCs, similar conditions used to induce chondrogenesis in MSCs ³¹ were employed for the ASC population ^36, 43. In optimizing ASC differentiation protocols, it was later discovered that other members of the transforming growth factor beta (TGF-β) superfamily, such as bone morphogenetic protein 6 (BMP-6), can serve as potent regulators of ASC chondrogenesis ^45, 48. In general, chondrogenesis requires a three-dimensional culture system, and ASCs can be successfully induced down a chondrogenic lineage in a variety of scaffold environments. These culture configurations include pellet culture, which takes advantage of cell-cell interactions in a similar fashion to condensation during cartilage development ⁴⁹, alginate bead culture which employs an inert hydrogel to facilitate a rounded cell phenotype advantageous for chondrogenesis ³⁶, and cartilage-derived matrix which seeks to recapitulate some of the cell-matrix interactions seen in native cartilage ^50, 51.

To repair or regenerate articular cartilage, an understanding of the molecular constituents and their role in the mechanical function of cartilage must be taken into consideration. Articular cartilage provides joint congruity and a lubricated surface for articulation, effectively distributing loads of up to ten times body weight that pass through the joint during normal physiologic activity ⁵². Remarkably, this tissue provides a nearly frictionless bearing surface for the joint and functions over decades of use with little or no wear under normal circumstances. Articular cartilage is > 60% collagen by dry weight ^52, 53, primarily consisting of collagen type II with lesser amounts of other collagens (e.g., Types VI, IX, X, and XI). The collagen fibrils are located throughout the matrix and are intertwined with a highly concentrated negatively charged proteoglycan matrix ^54, 55. Two primary glycosaminoglycans (GAG) are found in articular cartilage, chondroitin sulfate and keratin sulfate. GAG side chains (polymer repeats of the sulfated molecule) are found assembled covalently to a protein core to form a proteolgycan aggregate (reviewed in ⁵⁴). The large aggregating proteoglycan, aggrecan, is assembled into a complex structure by a noncovalent linkage of these proteoglycan aggregates to a hyaluronate backbone, producing immobilized structures contributing to the articular cartilage solid matrix ⁵³. The assembly of the cartilage matrix gives the tissue its unique set of biomechanical properties by virtue of the ability of the collagen-proteoglycan matrix together with the interstitial fluid to effectively resist high levels of stress and strain engendered by normal loading of the joint. While creating a true mimic of articular cartilage may not be necessary for proper function, a tissue possessing similar molecular constituents will most likely possess similar mechanical properties. Thus, monitoring the assembly and spatial organization of key extracellular matrix components is an important step in the development of tissue-engineered cartilage implants.

Markers of Chondrogenesis

Herein, we report the methods for ASC isolation and expansion, scaffold preparation, encapsulation of cells, and biochemical conditions to induce chondrogenesis. See Figure 1 for a flow chart showing the procedure and associated timing information. We further report methods used to evaluate the degree of chondrogenic differentiation in 3D culture (Table 1).

Figure 1

Flow chart for experiments with associated timing

Table 1

Assaysto determine ASC chondrogenesis

Controls

We have previously shown that only 43% of ASCs at a clonal level are capable of chondrogenic differentiation ³⁷. Because of this, it is relatively straightforward to select cells that have both high and low chondrogenic differentiation potential, which can be used for positive and negative controls respectively. Also, in most circumstances, ASCs can be cultured in a control medium without growth factors to serve as a negative control ^{45, 51, 56, 57}. Specifically, as relates to histology and immunohistochemistry, sections of articular cartilage should be used as positive controls to assess the degree of chondrogenic differentiation of the tissue engineered constructs.

Appropriate Selection of an ASC Culture System

In this protocol, we describe two commonly used 3D culture systems that can be employed to chondrogenically induce ASCs. While both of these culture systems have been successfully used for ASC chondrogenesis, the selection of one over the other depends highly on the study purposes. The use of pellet culture results in high spatial cell density and necessitates cell-cell contact, much like the cellular condensation process during limb development ⁴⁹. The use of alginate results in a rounded cell morphology, much like that observed in articular cartilage, which has also been shown to be an important factor in promoting ASC chondrogenesis ³⁶. The use of pellet culture mimics the development of cartilage during limb formation and is therefore often used as a method to understand the interaction of cells and growth and environmental factors to promote a chondrogenic phenotype ^{37, 48, 58, 59}. While alginate can also be used for this purpose ^{35, 36, 51, 56, 60}, the use of alginate and other hydrogels can have profound influences on the ensuing phenotype of the cells, ^35, 50, 61 and therefore, the effect of the biomaterial as a significant variable influencing the differentiation potential of the cells must be taken into account when choosing an appropriate culture system. It is also important to note that while we report on the methods for two often used cell culture systems, many other materials and culture systems have been reported for being supportive of ASC chondrogenesis ^35, 50, 62 and should also be considered when selecting an ASC culture system. Regardless of the culture system employed, the methods for inducing chondrogenesis with the growth factors listed in Table 3 may still serve as a common starting ground for ASC chondrogenesis.

Table 5

Growth Factor Combinations for ASC chondrogenesis*

MATERIALS

REAGENTS

Cell isolation and expansion

Adipose tissue (contains blood)

Caution. See note below step 4 for guidelines in dealing with human tissue.
5, 10, and 25 ml serological Pipettes, sterile (Corning, cat. Nos. 4487, 4488, and 4489 respectively)
250 ml plastic bottles for centrifuging, sterile (Corning, cat. No. CLS430236)
50 mL polypropylene centrifuge tubes, sterile, (Corning, cat. No. 430290)
2 1L beakers, sterile
10% (v/v) bleach

Caution. Corrosive. Causes eye, skin, and digestive tract burns. Harmful if inhaled and results in respiratory tract irritation. Use appropriate personal protective equipment to avoid exposure (i.e., wear gloves, safety glasses and a mask, or use material in fume hood.)
70% (v/v) ethanol

Caution Ethanol is highly flammable. May also cause eye and respiratory tract irritation. Use appropriate personal protective equipment to avoid exposure (i.e., wear gloves, safety glasses and a mask, or use material in fume hood.)
10% (v/v) dish washing detergent
Dulbecco’s phosphate-buffered saline (D-PBS), without calcium chloride and magnesium chloride (Gibco, cat. no. 14190-144)
BSA, Fraction V, 7.5% solution (Gibco, cat. No. 15260-037)
Type I Collagenase (Worthington Biochemical Products, cat. No. LS004197)

Critical: We advise testing several lots of collagenase to insure effective digestion of the adipose tissue. There is significant variability between lots provided by a single commercial vendor.
1 M calcium chloride solution (sterile)
250 ml filter 0.22 μm low protein binding sterilization unit (Corning, cat. no. 430767)
225cm² Cell Culture Flask (Corning, cat. no. 431082)
Dulbecco’s Modified Eagle Medium/Ham’s F-12 Nutrient Broth (1:1. v/v) with 15 mM HEPES buffer, L-glutamine and pyridoxine hydrochloride (Gibco, Cat. No. 11330-032 or Biowhittaker, Cat No. 12-719F)
Ammonium chloride (NH₄Cl) (Sigma, cat. No. A0171)
Potassium carbonate (K₂CO₃) (Sigma, cat. No. P5833)

Caution. Causes eye, skin, and respiratory tract irritation. Harmful if swallowed. Use appropriate personal protective equipment to avoid exposure (i.e., wear gloves, safety glasses and a mask, or use material in fume hood.)
Fetal Bovine Serum (Atlas Biologicals, Cat. No. F-0500-A)

Critical: We advise testing several lots of serum for culture optimization, and then use of the same lot of FBS for an entire series of experiments. The authors have observed significant variability in the results obtained with different sera.
0.05% Trypsin/EDTA (Gibco, Cat. No. 15140-122)
Penicillin/Streptomycin/Fungizone (Gibco, Cat. No. 15240-062)
Human epidermal growth factor (rhEGF) (Roche, Cat. No. 1376454)
Human fibroblastic growth factor, basic (rh-bFGF) (Roche, Cat. No. 1123149)
Transforming Growth Factor beta-1 TGF-β1 (R&D Systems, Cat. No. 100-B-001)
Cryogenic Vials (Corning, cat. No. 430488)
Dimethylsulfoxide (DMSO) Hybri-Max® (Sigma, Cat. No. D2650)

Caution Harmful if swallowed, inhaled, or absorbed through skin. Causes eye, skin, and respiratory tract irritation. Use appropriate personal protective equipment to avoid exposure (i.e., wear gloves, safety glasses and a mask, or use material in fume hood.)
1°C Freezing Container (Nalgene, Cat. No. 5100-0001))

Alginate bead culture

Alginate (Pronova^™ LVG USP, Cat. No. 4200001)
Sodium Chloride (NaCl) (Sigma, Cat. No. S9625)
Sodium Citrate Trisodium salt dihydrate (Sigma, Cat. No. S4641)
Calcium Chloride (Sigma, Cat. No. C3306)
Sterile Syringe filter (0.22 mm) (VWR International, Cat. No. 28145-477)
150 ml 0.22 mm filter system Corning, Cat. No. 431154).
24 well plate, with lid, flat bottom, Ultra-low attachment surface (Corning, Cat. No. 29443-032)
VWR Spinbar® Micro stir bars (12.7 mm × 3 mm) (VWR International, Cat. No. 58948-397)

Chondrogenic Induction

Dulbecco’s Modified Eagles Medium-high glucose, (DMEM-HG), (Gibco, Cat. No. 11995-065)
ITS+ supplement, (Collaborative Research, Cat. No. 40352)
Dexamethasone, (Sigma, Cat. No. D-4032)
L-Ascorbic acid 2-phosphate Sesquimagnesium Salt (Sigma, Cat. No. A8960)
Penicillin/Streptomycin (Gibco, cat. No. 15140-122)
Transforming Growth Factor beta-3 (TGF-β3), (R&D Systems, Cat. No. 243-B3-002)
Bone Morphogenetic Protein-6 (BMP-6), (R&D Systems, Cat. No. 507-BP-020)
Siliconized 200 μl Pipette tips (VWR, Cat. No. 53503-792)
Siliconized 0.6 mL Snap-Cap microtubes (Sigma, Cat. No. T4691-500EA)
15 mL polypropylene centrifuge tubes, sterile, (Corning, cat. No. 430052)
50 mL polypropylene centrifuge tubes, sterile, (Corning, cat. No. 430290)

Papain Digestion solution

L-Cysteine Hydrochloride Anhydrous (Sigma, Cat. No. C1276)

Caution. Avoid contact. Causes eye, skin, and respiratory tract irritation. Use appropriate personal protective equipment to avoid exposure (i.e., wear gloves, safety glasses and a mask, or use material in fume hood.)
Ethylenediaminetetraacetic acid, EDTA (Sigma, Cat. No. EDS-100G)

Caution. Avoid contact. Causes eye, skin, and respiratory tract irritation. Use appropriate personal protective equipment to avoid exposure (i.e., wear gloves, safety glasses and a mask, or use material in fume hood.)
Papain, 0.5 U/mg (Sigma, Cat. No. 76222)

Caution. Avoid contact. Causes eye, skin, and respiratory tract irritation. Use appropriate personal protective equipment to avoid exposure (i.e., wear gloves, safety glasses and a mask, or use material in fume hood.)
Sodium phosphate monobasic (Mallinckrodt, Cat. No. 7892)

Caution. Avoid contact. Causes eye, skin, and respiratory tract irritation. Use appropriate personal protective equipment to avoid exposure (i.e., wear gloves, safety glasses and a mask, or use material in fume hood.)

dsDNA Quantitation

PicoGreen dsDNA Quantitation kit (Invitrogen, Cat. No. P-7589)

DMB Assay

Sodium Formate (Sigma, Cat. no. S2140)

Caution. Harmful if swallowed or inhaled. Causes eye, skin, and respiratory tract irritation. Use appropriate personal protective equipment to avoid exposure (i.e., wear gloves, safety glasses and a mask, or use material in fume hood.)
1,9-Dimethyl-Methylene Blue (Sigma, Cat # 341088, Sigma)

Caution. Known eye irritant. Use appropriate personal protective equipment to avoid exposure (i.e., wear gloves and safety glasses and allow for adequate ventilation.)
Chondroitin 4-sulfate (C-4-S, type A – from Bovine Trachea) (Calbiochem, Cat. No. 230687)
Formic Acid (EM Science, Cat. No. FX 0440-7)

Caution. Harmful if swallowed or inhaled. Causes eye, skin, and respiratory tract irritation. Use appropriate personal protective equipment to avoid exposure (i.e., wear gloves, safety glasses and a mask, or use material in fume hood.)
Clear, flat-bottom 96-well plate (BD, Cat. No. 353228)

Hydroxyproline assay

Chloramine-T, hydrate 98% (Sigma, Cat. No. 857319)

Caution. Harmful if inhaled. Substance is known to be destructive to the tissue of the mucous membranes and upper respiratory tract. Also causes eye and skin burns and may be harmful if swallowed. Use appropriate personal protective equipment to avoid exposure (i.e., wear gloves, safety glasses and a mask, or use material in fume hood.)
n-Propyl alcohol 1 L (Sigma, Cat. No. P6334)

Caution. n-Propyl alcohol is highly flammable. Harmful if swallowed or inhaled or absorbed through skin. Use appropriate personal protective equipment to avoid exposure (i.e., wear gloves, safety glasses and a mask, or use material in fume hood.)
p-Dimethylaminobenzaldehyde (Sigma, Cat. No. D8904)
Perchloric Acid 60% 1 lb (Fisher Scientific, Cat. No. A228)

Caution. Harmful if swallowed or inhaled. Strong oxidizer. Corrosive. Use appropriate personal protective equipment to avoid exposure (i.e., wear gloves, safety glasses and a mask, or use material in fume hood that is not designed for other uses.)
Citric Acid Monohydrate (Sigma, Cat. No. C1909)

Caution. Known eye irritant; results in severe eye irritation and possible injury. Also causes skin and respiratory tract irritation. Use appropriate personal protective equipment to avoid exposure (i.e., wear gloves, safety glasses and a mask, or use material in fume hood.)
Sodium acetate Trihydrate (Sigma, Cat. No. S9513)
Sodium Hydroxide Pellets (Mallinckrodt, Cat. No. 7708)

Caution. Highly corrosive. Can result in eye and skin burns. May also result in respiratory and/or digestive tract irritation with possible burns. Use appropriate personal protective equipment to avoid exposure (i.e., wear gloves, safety glasses and a mask, or use material in fume hood.)
Acetic Acid Glacial (EM Science, Cat. No. UN 2789)

Caution. Highly corrosive and flammable. Can result in severe burns to all body tissue and may be fatal if swallowed. Inhalation may cause lung and tooth damage. Use appropriate personal protective equipment to avoid exposure (i.e., wear gloves, safety glasses and a mask, or use material in fume hood.)
Hydrochloric Acid, 37% (6.0 N) (EM Science, Cat. No. UN1789)

Caution. Highly corrosive. Can result in severe burns to all body tissue and may be fatal if swallowed or inhaled. Inhalation may cause lung damage. Use appropriate personal protective equipment to avoid exposure (i.e., wear gloves, safety glasses and a mask, or use material in fume hood.)
trans-4 hydroxyproline-L (Sigma, Cat. No. H54409)
Activated Charcoal Powder (EM Science, Cat. No. CX0645-1)
1.5 ml microcentrifuge tubes (VWR, Cat. No. 20170-038)
Costar Spin-X HPLC micro centrifuge tube (nonsterile) and filter (0.45 μm nylon filter) (Corning, Cat. No. 8170)

Fixation

16% paraformaldehyde (Electron Microscopy Sciences, Cat. No. 15710)

Caution. Paraformaldehyde is a suspected carcinogen. Avoid contact and inhalation. Use appropriate personal protective equipment to avoid exposure (i.e., wear gloves, safety glasses and a mask, or use material in fume hood.)
Sodium Cacodylate, Trihydrate (Electron Microscopy Sciences, Cat. No. 12300)

Caution. May be fatal if swallowed or inhaled. Harmful if absorbed through skin. Contains arsenic which can cause cancer. Skin, eye, and respiratory tract irritant. (i.e., wear gloves, safety glasses and a mask, or use material in fume hood.)
Barium chloride (Mallinckrodt, Cat. No. 3756)

Caution. Known irritant. May cause eye, skin, and respiratory tract irritation. May be fatal if swallowed or inhaled. Avoid contact and inhalation. Use appropriate personal protective equipment to avoid exposure (i.e., wear gloves, safety glasses and a mask, or use material in fume hood.)

Histology

Xylene (Mallinckrodt, Cat. No. 8668-16)

Caution. Harmful or fatal if swallowed. Causes severe eye irritation. Use appropriate personal protective equipment to avoid exposure (i.e., wear gloves, safety glasses and a mask, or use material in fume hood.)
Paraplast® Tissue Embedding Medium (Fisher Scientific, Cat. No. 23-021-399)

Caution Paraffin burns readily. Keep away from fire.
Tissue-Tek® Uni-Cassette® LWS (Sakura, Cat. No. 4156-02)
Fisherbrand® Superfrost®/Plus Microscope Slides (Fisher Scientific, Cat. No. 12-550-15)
Safranin-O (Sigma, Cat. No. HT90432-1L)

Caution. Causes eye, skin, and respiratory tract irritation. Avoid contact and inhalation. Use appropriate personal protective equipment to avoid exposure (i.e., wear gloves, safety glasses and a mask).
Toluidine blue (Sigma-Aldrich, cat. no. 89640-5G)

Caution May cause gastrointestinal and blood disorders. Avoid contact and inhalation. Use appropriate personal protective equipment to avoid exposure (i.e., wear gloves, safety glasses and a mask, or use material in fume hood.)
Fast Green FCF (Sigma, Cat. No. XXXXF7252-5G)

Caution May be harmful if swallowed or inhaled. May cause eye, skin, and respiratory tract irritation. Avoid contact and inhalation. Use appropriate personal protective equipment to avoid exposure (i.e., wear gloves, safety glasses and a mask, or use material in fume hood.)
Weigert Hematoxylin Solution (Sigma, Cat. No. HT 1079)

Caution. Known irritant. May cause eye, skin, and respiratory tract irritation. Avoid contact and inhalation. Use appropriate personal protective equipment to avoid exposure (i.e., wear gloves, safety glasses and a mask, or use material in fume hood.)
Differentiation solution (Sigma, Cat. No. A3179)

Caution. Corrosive. Flammable; keep away from fire. Avoid prolonged exposure. Use appropriate personal protective equipment to avoid exposure (i.e., wear gloves, safety glasses and a mask, or use material in fume hood.)
Permount® (Fisher Scientific, Cat. No. SP15-100)

Caution. Contains toluene. Causes eye, skin, and respiratory tract irritation. Avoid contact and inhalation. Inhalation may cause drowsiness and dizziness. May cause central nervous system depression. Use appropriate personal protective equipment to avoid exposure (i.e., wear gloves, safety glasses and a mask, or use material in fume hood.)

Immunohistochemistry

Zymed Histostain® Plus Broad Spectrum (Invitrogen, Cat. No. 85-8943)
Type II collagen antibody (Developmental Studies Hybridoma Bank, Cat. No. II-II6B3 ordered as supernatant)
Type I collagen antibody (AbCam, Cat. No. AB6308)
Type X collagen antibody (Sigma, Cat. No. C7974)
2-B-6 Chondroitin-4-Sulfate antibody (Seikagaku, Cat. No. 270432)
3-B-3 Chondroitin-6-Sulfate antibody (Seikagaku, Cat. No. 270433)
Anti-Mouse IgG (Fab specific)–Biotin secondary antibody produced in goat (Sigma, Cat. No. B7151)
Xylene

Caution. Harmful or fatal if swallowed. Causes severe eye irritation. Use appropriate personal protective equipment to avoid exposure (i.e., wear gloves, safety glasses and a mask, or use material in fume hood.)
AEC Substrate-Chromagen (Invitrogen, Cat. No. 00-1111)
Digest-All^™ 3 (Pepsin) (Invitrogen, Cat. No. 00-3009)
Chondroitinase ABC (Sigma, Cat. No. C 2905)
PAP Pen (Research Products International, Cat. No. 195505)
Methanol (EMD, Cat. No. MX0485-7)

Caution. Harmful if swallowed. Highly flammable; keep away from heat and ignition sources. Use appropriate personal protective equipment to avoid exposure (i.e., wear gloves, safety glasses and a mask, or use material in fume hood.)
Hydrogen Peroxide (EMD, Cat. No. HX0635-2)
Goat serum (Invitrogen, Cat. No. 50-062Z)
GVA Mounting Medium (Invitrogen, Cat. No. 008000)

qPCR

RNASecure^™ Reagent (Applied Biosystems, Cat. No. AM7005)
2 mL microcentrifuge tubes (RNase/DNase free) (VWR, Cat. No. 87003-298) Note: the design of these microcentrifuge tubes (wider bottom) allows more efficient chelation of alginate.
Table 2: TaqMan Assays on Demand for assessing chondrogenesis ⁴⁵

Table 2

TaqMan Assays on Demand

Equipment

Centrifuge
Water bath shaker
Microplate reader (for fluorescence and absorbance based assays)
Speedvac or lyophilizer
Microtome
Real-Time PCR Instrument
Hot plate with temperature control

REAGENT SETUP

Cell Isolation

Expansion medium: Use all reagents listed in Table 1. Expansion medium may be stored at 4° C in the dark for up to 2 weeks.

Table 3

Media components for expanding ASCs

Stromal medium: Use all reagents listed in Table 1, except for rhEGF, rhFGF, and TGF-β1 (i.e., stromal medium should not contain any growth factors). Stromal medium may be stored at 4° C in the dark for up to 6 weeks.

Collagenase solution: This should be made fresh before starting the isolation and filter sterilized using a 250 ml sterilization unit. Make a 1% (v/v) solution of BSA in D-PBS. To this solution, add Type I collagenase to make a 0.1% (w/v) collagenase solution and 1 M calcium chloride to a final concentration of 2 mM.

Red cell lysis buffer: Prepare a sterile solution of 155 mM ammonium chloride (NH₄Cl), 10 mM potassium carbonate (K₂CO₃), and 0.1 mM EDTA. Use within two weeks of preparation.

Freeze medium: Make a solution containing 80% (v/v) FBS, 10% (v/v) DMEM/F12, and 10% (v/v) DMSO.

Cell Differentiation

Incomplete chondrogenic medium. Make up using all reagents listed in Table 2. This medium may be stored at 4° C in the dark for up to 6 weeks.

Table 4

Incomplete chondrogenic medium

Complete chondrogenic medium: Thaw growth factors and add freshly to incomplete chondrogenic medium just before use. Three different growth factor combinations have been used successfully to promote ASC chondrogenesis (Table 3).

Alginate Culture

Preparation of 1.2% (w/v) alginate solution: Dissolve 1.2 g of alginate in 100 ml of 150 mM NaCl. Heat on a hot plate and stir thoroughly. Filter sterilize using a 0.22 μm filter. Another option is to prepare the solution using aseptic technique in a sterile hood, thus obviating the need for filter sterilization. Store resulting alginate solution (1.2%) at 4°C.

CaCl₂ (102 mM) solution: Place 10.2 ml of a 1 M CaCl₂ stock solution into 100 mL volumetric flask and then pipette in 89.8 mL dH₂O. Filter through 250 ml filter system. Store working solution (102 mM) at 4°C.

NaCl (150 mM) solution: Place 15.0 ml of the 1 M NaCl stock solution into 100 mL volumetric flask and then pipette in 85 mL dH₂O. Filter through 250 ml filter system. Store working solution (150 mM) at 4°C.

Sodium Chloride (50 mM) – Sodium Citrate (55 mM) buffer: Dissolve 1.461 g of NaCl into 300 ml of dH₂0. Dissolve 8.087 g of sodium citrate into this same solution. Add dH₂0 to a final volume of 500 ml.

Sample fixative for histology/immunohistochemistry

Pipette 10 ml of 16% paraformaldehyde into a 50 ml conical tube. Dissolve 0.856 g of sodium cacodylate. If alginate is being fixed, add 0.104 g BaCl₂ to irreversibly cross-link the alginate matrix. Titrate the solution with 1 N HCl to a pH of 7.4. Adjust the final volume of the solution to 40 ml with dH₂0. Caution: paraformaldehyde and sodium cacodylate are considered carcinogenic. Only handle these reagents in a biosafety level 3 cabinet with the sash adjusted to the proper height to provide maximum protection to the user.

DMB Assay

Preparation of 1,9-dimethlemethylene blue (DMB) dye: Dissolve 21 mg of 1,9-dimethtlemethylene blue into 5 ml of absolute ethanol with 2.0 g of sodium formate and stir thoroughly in 800 ml of distilled water. Titrate concentrated formic acid into the dye solution to adjust the pH to the desired level (pH 1.5 for the alginate DMB assay and pH 3.0 for the pellet cultures). Add distilled water to make a final volume of 1000 ml. This solution may be stored for up to 6 months at room temperature (RT, 19 to 25°C). Note that the solution should be protected from light.

Preparation of Stock Chondroitin-4-Sulfate Standard Solution (10 mg/ml): Weigh 100 mg of chondroitin-4-sulfate (C-4-S). Dissolve the powder in 10 ml of D-PBS in a 50 ml conical tube. Vortex to insure thorough mixing. Dilute 1 ml of the Stock C-4-S solution into 9 ml of Papain solution. This produces a 1 mg/ml C-4-S solution. Freeze in 1 ml aliquots and store at −80°C. Thaw an aliquot on the day of the assay to prepare the standard curve for the DMB assay.

Hydroxyproline assay

Acetate-Citrate Buffer pH 6.5Dissolve the 12 g of Sodium acetate trihydrate, 5 g of citric acid monohydrate, 1.2 mL acetic acid, and 3.4 g sodium hydroxide in 750 ml of dH₂O. Measure pH and adjust to 6.0 if necessary by titration with 1 N NaOH or acetic acid. Bring the solution to a final volume of 1000 ml with dH₂O. Can be prepared and stored for 1 – 2 months at room temperature.

Chloramine-T Reagent (0.062 M), Dissolve 141 mg of chloramine-T in 2.07 ml of dH₂O and 2.6 ml n-propanol. Note: mix dH₂O and n-propanol first. Add 5.33 ml of the Acetate-Citrate buffer described above. Vortex to dissolve completely. CRITICAL: Must be prepared fresh just before it is to be used.

p-DMBA Reagent (0.94 M), Dissolve 1.4 g of p-dimethylaminobenzaldehyde in 7 ml of n-propanol and vortex. Add 3.0 ml of perchloric acid. Vortex to dissolve completely. CRITICAL: Must be prepared fresh just before it is to be used and used within 1 hour.

Hydroxyproline Standard stock (10 mg/ml), Dissolve 100 mg of trans-4 hydroxyproline-L in 10 mL of distilled water to get a stock solution of 10mg/mL. Vortex to dissolve completely. Can be made in advance and stored in 1 ml aliquots at −80°C.

PROCEDURE

ASC Isolation

1. Prepare the water bath shaker at 37 °C, with enough water to cover the bottles containing collagenase mixture.
2. Prepare fresh a solution of 0.1% (w/v) collagenase, 1 % (v/v) bovine serum albumin (fraction V), and 2 mM calcium chloride in PBS. The volume of the collagenase solution should equal the volume of lipoaspirate to be processed (usually 100 to 200 ml).
3. Warm the D-PBS, collagenase solution, and medium to 37 °C.
4. Allow the fat transport container to sit until the fat and blood are well separated. Transfer the fat to a sterile, 250 ml plastic centrifuge tube for separation. Note: cell isolations are routinely performed the day of receipt or performed the next day, after leaving the tissue at room temperature overnight.

CAUTION: Procedures must be performed in accordance with Institutional Review Board policies for obtaining human tissue including informed consent by personnel certified and trained to work with blood borne pathogens. All procedures involving the human tissue should be performed at Biosafety Level 2 with appropriate personal protection.
5. Using a 25 ml serological pipette, aspirate off the blood layer from beneath the floating fat.
6. Check the tissue volume; it should be approximately 100 ml in each 250 ml centrifuge tube.

The isolated cells will be plated at an equivalent to ~35 ml of liposuction aspirate digest per T225 flask so plan the amount of tissue needed accordingly. If there is less tissue volume, the flask size should be reduced accordingly to achieve approximately 0.16 ml tissue per square centimeter.
7. Wash the fat with D-PBS (1:1 volume) up to 7 times or until the color of the D-PBS layer is about the hue of the fat – a light pink color. Allow the two phases to separate between washes (3–5 min). Gently stir the solution while it sits; this will encourage separation and aid the washing. Continue to remove the blood layer from beneath the yellow fat tissue. After a few washes, a bright yellow layer of extra cellular fat will begin to form on top of the fat cell layer. Carefully aspirate off this thin layer. Note that 4 washes should be sufficient.
8. After the fat layer has been thoroughly washed, mix it 1:1 (v:v) with the warm D-PBS:collagenase solution. Place it in the 37° C shaker water bath and gently shake for 1 hour. Shake to mix by hand every 20 minutes if using 250 ml centrifuge bottles; or shake to mix by hand every 5–10 minutes if using 50 ml centrifuge tubes.

Note that if a shaker water bath is not available, 50 ml or 250 ml tubes may be placed on a shaker in a 37° C incubator. TROUBLESHOOTING
9. Centrifuge at 300 g at 21° C for 5 minutes.
10. Shake the tubes vigorously for 10 seconds to ensure that individual cells are released from the strands of fibrous tissue.
11. Centrifuge at 300 g at 21° C for 5 minutes.
12. Aspirate off the floating mature adipocyte layer and the aqueous collagenase/D-PBS supernatants, leaving 5 ml of D-PBS on each pellet of cells. Be careful with the cell pellet as it is gelatinous and does not adhere readily to the centrifuge tubes. TROUBLESHOOTING
13. Resuspend each cell pellet in 10 ml stromal medium and pool the suspensions into sterile, 50 ml centrifuge tubes.
14. Centrifuge at 300 g at 21° C for 5 minutes.
15. Aspirate off the supernatants, leaving 5 ml of stromal medium on each pellet.
16. Resuspend cell pellets in 10 ml stromal medium and pool contents into a sterile, 50 ml centrifuge tubes.
17. Centrifuge at 300 g at 21° C for 5 minutes.
18. Aspirate off the supernatants, leaving 5 ml of stromal medium on each pellet.

Note that the pellet can look very different from patient to patient at this stage.
19. Resuspend in a total of 105 ml stromal media per 100 ml of lipoaspirate digest.

TROUBLESHOOTING

ASC Expansion

20. Plate each dish at an equivalent density to 0.16 ml of liposuction aspirate per cm² or ~35 ml per 225cm² flask.
21. Thoroughly clean up by collecting all contaminated materials into biohazard bags.
22. Clean the hood with 10% detergent, then 10% bleach, then 70% ethanol.
23. After 24 hours, change the media on the cells to wash away any non-adherent cells.
24. Feed the cells every three days for up to 7 days by replacing 100% of the media in each flask.

TROUBLESHOOTING
25. Harvest cells when they are 80% confluent by trypsinization. Aspirate off expansion medium. Wash the flasks gently with 20 ml of D-PBS warmed to 37° C. Add 8 ml of trypsin/T225 flask, and incubate for 5 minutes at 37 ° C. Verify that the cells have been dislodged from the cell culture plate using a microscope. Inactivate trypsin with an equal amount of stromal medium (i.e., 8 ml).
26. Collect the 16 ml of trypsin/stromal medium in a 50 ml conical tube.
27. Centrifuge at 300 g at 21° C for 5 minutes to pellet cells. Aspirate medium on top of cells.
28. Either passage ASCs further (option A), freeze cells (option B), proceed with chondrogenic differentiation in pellet culture (option C) or proceed with chondrogenic differentiation in alginate beads (option D).

Option A. Further passage. TROUBLESHOOTING

Resuspend ASCs in expansion medium and replate at 8,000 cells/cm². Representative images of ASCs through 1 passage can be seen in Figure 2. CRITICAL cells may be used up to passage 4, after which the cells will start to lose their differentiation potential.

Figure 2

Expansion of ASCs in expansion medium over 5 days. Appearance of cells at 10x after (a) 30 minutes, (b) 3 hours, (c) 1 day, (d) 2 days, (e) 3 days, (f) 4 days, and (g) 5 days. Black arrows in (h) and (i) show cells with atypical or abnormal morphology. …
Ii)Trypisinize cells at ~80% confluence (Figure 2g).

Option B. Freeze cells.

Make aliquots of 1–5 million cells/ml in freeze medium in cryovials.
Freeze cells in freeze medium at a rate of −1° C/min using a 1°C freezing container until they reach −80° C, after which the cells may be stored long-term in liquid nitrogen.

PAUSEPOINT Cells can be stored long-term in liquid nitrogen. When thawing cells, place vial in 37° C water bath to thaw cells rapidly. Immediately upon thawing, remove cells from vial and wash cells in 2–3 ml of stromal medium. Centrifuge at 300 g at 21 °C for 5 minutes after which the cells can be resuspended and plated at 8,000 cells/cm².

Option C. Chondrogenic differentiation in pellet culture:

Following trypsinization of the cells, split desired number of cells (250,000 per pellet) to 15 ml tubes designated for either negative control or chondrogenic conditions.
Resuspend cells in either Incomplete (control) or Complete Chondrogenic Medium at a density of 500,000 cells/mL. Incomplete chondrogenic medium can be used as a negative control. Complete chondrogenic medium with ASCs known to be capable of chondrogenesis may be used as a positive control.
Pipette 0.5 ml of cell suspension into 15 mL polypropylene conical tubes. This yields 250,000 ASCs per tube.
Centrifuge at 300 g at 21 °C for 5 minutes to form a pellet at the bottom of the tube.

Loosen the tops of the conical tubes for gas exchange. Incubate cultures at 37°C and 5% CO₂overnight.

The following day, the pellets should be rounded in the bottom of the tube.
Every other day, for the duration of the experiment, prepare complete chondrogenic medium by adding growth factors and L-Ascorbic acid 2-phosphate and exchange medium. Typical durations for culture are 2, 4, and 6 weeks.
At each medium exchange, agitate tube gently to ensure the pellet has not adhered to the wall of the tubes.
To harvest, remove the chondrogenic medium and wash the pellets once with D-PBS. Fix the pellet in the paraformaldehyde solution for immunohistochemistry and histology, or digest the pellet in a papain solution for biochemical analysis (see step 29).

Option D. Chondrogenic differentiation in alginate beads:

Warm alginate and CaCl₂ to 37 °C prior to encapsulation of ASCs.
Resuspend ASCs in 1.2% alginate solution at 5×10⁶ cells/ml in a 50 ml conical tube. Mix thoroughly by pipetting without creating bubbles, or mix with the use of micro stir bars using a magnetic stirring plate. Note that if the latter technique is used for mixing, a volume of greater than 700 μl is required to avoid making bubbles.
Using a 1 ml pipette, draw 1 ml of solution into tip.
Dispense by tilting the pipette sideways and slowly pipetting cell suspension such that one drop falls from the pipette tip into 1 ml of pre-warmed CaCl₂. Typically, 3 drops of alginate are added to each well of a low attachment surface 24 well plate. (Figure 3 shows a 2.5x image of ASCs encapsulated within alginate.) Note that alginate bead diameter will vary from ~4 – 5mm and will contain approximately 275,000 cells. Five to six wells can be filled per ml of alginate.

Figure 3

2.5x microscope image (2.5x) of ASCs encapsulated in alginate. Scale bar = 1 mm.
Incubate the alginate beads at 37°C for 5 minutes to allow Ca⁺² cations to fully diffuse through the alginate and cross-link the alginate cell suspension.
Pipette off the CaCl₂ solution. Note that aspiration can be used, but care must be taken to avoid suction pressure on the alginate beads.
Wash the beads with 1.5 ml incomplete chondrogenic medium at 37°C for 15 minutes. Pipette off incomplete medium and repeat for an additional 15 minutes at 37°C.
Replace incomplete medium with complete chondrogenic medium, 1 ml/well of a 24 well plate. Note that typically 1 ml of medium is used for every 800,000 to 1,000,000 cells. Incomplete chondrogenic medium can be used as a negative control. Complete chondrogenic medium with ASCs known to be capable of chondrogenesis may be used as a positive control.
Incubate at 37°C, 5.0% CO₂. According to experimental design, take samples at day 0 and subsequent time points to be prepared for biochemical examination, histological and immunohistological examination, or gene expression analysis. Accordingly, proceed to the applicable section of this protocol. Refer to Figure 1 for timing for procedures. Every other day for the duration of the experiment, prepare complete chondrogenic medium by adding growth factors and L-Ascorbic acid 2-phosphate and exchange medium. Typical durations for culture are 2, 4, and 6 weeks.

Biochemical determination methods

29

There are a number of options as to how to process the cells further. See Table 1 for information about what each assay assesses. If you wish to perform the dimethyl-methylene blue (DMB) assay^34, 35, follow option A. If you wish to do the hydroxyproline assay^35, 64, 65, follow option B. If you wish to perform histology or immunohistochemistry proceed with option C for all samples followed by option D for safranin-o/fast green staining, option E for toluidine blue staining or option F for immunohistochemistry. If you wish to do RT-PCR follow option G.

Option A) Dimethylmethylene blue (DMB) Assay

Harvest samples at appropriate time points and digest in 1 ml of papain solution (for alginate beads, 3 beads/well = 1 construct) for 15–18 hours at 65°C. CRITICAL: If wet weight is desired for normalization, weigh the samples before digestion in papain.

PAUSEPOINT If desired, digested samples can be stored at −20°C and then thawed for analysis.
Determine total DNA per construct using the PicoGreen dsDNA quantitation kit per the instructions from the manufacturer. Total dsDNA will be used to normalize DMB content.
Prepare the diluted standard solutions of C-4-S (from 0 to 35 μg/ml) mixing the quantities shown in Table 4 (in microliters) of the 1 mg/ml C-4-S solution with the Papain:

Table 6

Volumes for chondroitin 4-sulfate (C-4-S) standard curve
In a clear, flat-bottom 96-well plate, aliquot 40μl of the C-4-S standard solutions and samples to wells. Note that it is likely that some or all samples will need to be diluted with papain at this stage in order for the samples to fall in the range of the standard curve.
Add 125 μl of the DMB dye (pH 3.0 for samples not containing alginate) to each well of the plate using a multichannel pipette.
Measure the optical density of the solutions (standard and experimental) using the 595 nm filter (OD₅₉₅) on a plate reader. Follow option B step
Xxiii onward to calculate S-GAG content.

Option B) Hydroxyproline (OHP) Assay

Harvest samples at appropriate time points and digest in 1 ml of papain solution (for alginate beads, 3 beads/well = 1 construct) for 15–18 hours at 65°C. CRITICAL: If wet weight is desired for normalization, weigh the samples before digestion in papain.

PAUSEPOINT If desired, digested samples can be stored at −20°C and then thawed for analysis.
Determine total DNA per construct using the PicoGreen dsDNA quantitation kit per the instructions from the manufacturer. Total dsDNA will be used to normalize hydroxyproline content.
Using pre labeled test tubes, make up your standard solutions by mixing the following proportions of the 1 mg/ml working hydroxyproline solution with D-PBS as follows:
Aliquot 50 μl of the standard solutions and samples in prelabeled microcentrifuge tubes. Note that any dilutions that need to be carried out to bring samples within the range of the standard curve should be performed at this step using papain.
Add 50 μl of the 12 N HCl (37%). (Final Concentration ~6N)

CRITICAL: ensure caps close cleanly without deformation of plastic. Any deformation will result in evaporation of liquid before samples are hydrolyzed.
Hydrolyze the samples by incubating in an oven at 110°C for 15–18 hours (overnight).

TROUBLESHOOTING
Retrieve the tubes from oven.
Spin for 10 or 15 seconds in a microcentrifuge to collect any condensate on the sides and cap of the tubes.
Dry the samples completely either by lyophilization or by a Speedvac. Note that this could take several hours depending on the drying method used. Alternatively, one can remove the caps and place the samples in a laminar flow hood for 2–3 days to dry the samples.
Reconstitute the dried samples and standard in 100 μl of the Acetate-Citrate buffer. Mix thoroughly and vortex to dissolve completely.
Add enough activated charcoal to cover the filter chamber of the Spin-X HPLC column. Note that a 1 ml filter tip pipette works well for this purpose.
Place the reconstituted samples and standards in the filter chamber of the Spin-X HPLC microcentrifuge tubes. CRITICAL: Do not discard the tubes as they will be used again in step Xiv
Spin the samples for 3 minutes at 12,900 g in a microcentrifuge to filter the samples thorough the activated charcoal.
Add another 100 μl of the Acetate-Citrate buffer to the original tubes (now empty) to collect and reconstitute any residual amounts of the sample. Mix and vortex as before.
Place the reconstituted “residual” samples and standards in the filter chamber of the Spin-X HPLC microcentrifuge tubes.
Spin again for 3 minutes at 12,900 g in a microcentrifuge to filter the samples thorough the activated charcoal.
Aliquot triplicates of 50 μl of the samples and standards in individual wells of a 96 well plate.
Add 50 μl of Chloramine-T (0.062 M, prepared fresh as previously described) to the wells. Mix gently on an orbital shaker. Allow the oxidation to proceed for 15 minutes at room temperature.
For chromophore development, add 50 μl of the p-DMBA reagent to each sample.
Mix gently on an orbital shaker.
Allow the reddish/purple color development by incubating the samples at 37°C for 30 minutes.
Measure the optical density (OD) at 550 (or 540) nm on a plate reader.
Calculation of the amount of S-GAG or OH-proline: First calculate the average values of the duplicates or triplicates of each of the standard and experimental samples. Use these averages in the following calculations.
Calculate the “corrected” values of standard optical densities (OD) by taking the difference between the OD for the 0 μg/ml standard and that of the measured standard. Note that because DMB has a decreasing absorbance with increasing GAG concentration while OHP has increasing absorbance with increasing proline concentration, the terms in the following equations are switched to achieve positive values.
Calculate the “corrected” values of experimental samples optical densities (OD) by taking the difference between the OD for the 0 μg/ml standard and that of the measured samples
Plot the corrected optical density (x-axis) of each of the standard solutions versus the concentration (y-axis) of each of the standard solutions. Use linear regression (forcing a fit through the origin) to obtain the equation describing the linear relationship between the optical density and the concentration.
Using linear regression, calculate the concentration of sulfated glycosaminoglycans (GAGs)/OH-proline in the experimental samples based on the corrected OD_Sample values. Multiply the calculated concentration by the original sample volume and any dilution factors that were used.
For the OH-proline assay, determine the collagen concentration by using the conversion factor of 7.46 mg collagen to 1 mg 4-hydroxyproline. Note that a conversion factor of 10 mg collagen to 1 mg 4-hydroxyproline may be used instead of 7.46 to account for the presence of mostly type II collagen in constructs ⁶⁶.
Normalize the results to wet weight of constructs and/or total DNA of constructs. If samples are out of the standard range, dilute the samples appropriately and repeat the measurement, or prepare another standard spanning a wider range.

Option C) Histologic and immunohistochemical methods

Immediately following the culture period, place each construct (3 beads = 1 construct for alginate beads) in 20 ml of the paraformaldehyde solution. Fix for 4 hours at RT or overnight at 4°C.
Dehydrate constructs with 30% (v/v) EtOH (diluted in diH₂O) for 30 min, 50% EtOH for 30 min and70% EtOH for 30 min PAUSEPOINT Can be stored long term in 70% ethanol if not continuing immediately.
Dehydrate constructs with 80% EtOH for 30 min, 100% EtOH for 30 min, followed by one additional 100% EtOH wash. PAUSEPOINT. Can be stored overnight in 100% EtOH.
Clear constructs by removing 50% of solution and replacing with xylene yielding a final concentration of 50% EtOH/50% xylene. Incubate 30 min at RT. Replace this 1:1 mixture with 100% xylene and incubate 30 min at RT. Exchange 100% xylene and incubate for an additional 30 min at RT. CRITICAL: ensure that constructs are translucent. If not clear, continue processing with xylene washes until constructs become clear to translucent.
Embed the constructs in paraffin by removing half of the xylene and replacing it with paraffin such that the final concentration is 50% xylene/50% paraffin. Incubate at 60° C for 1 hr. Replace 1:1 mixture with 100% paraffin and incubate at 60° C for 1 hr; replace paraffin again with 100% paraffin and incubate again at 60° C for 1 hr.

CRITICAL: work quickly with the paraffin to ensure that the paraffin does not have time to solidify. If the paraffin solidifies, incubation times must increase to properly infiltrate the construct with paraffin.
Place in embedding tray in desired orientation (attempt to get all 3 alginate beads on the same plane for subsequent sectioning) and allow to harden overnight.
Cut sections 6–10 μm in thickness with a microtome and place in a 45–50° C waterbath.
Place sections on SuperfrostR/Plus Microscope Slides by using slides to remove from waterbath. Allow slides to dry overnight in a 37° C slide warmer. CRITICAL We advise using these particular slides as they have been surface treated for improved adherance of tissue section to slide during processing.

Option D) Safranin-O/fast green

Deparaffinize sections in xylene 3 times for 3 minutes each. (Note that for safranin-o/fast green staining, bone, muscles (collagen) are stained green, and cartilage is stained orange or red.)
Re-hydrate in 100% EtOH 2 × 5 minutes, 95% EtOH 1 × 2 minutes, and 70% EtOH 1 × 2 minutes.
Wash in tap water for 30 sec.
Stain in Weigert hematoxylin solution for 8 minutes.
Wash in tap water for 3 minutes by repeatedly dipping slides.
Differentiate in Differentiation Solution for 30 seconds.
Wash in tap water for 10 minutes by repeatedly dipping slides (until sections turn a blue hue)
Stain in 0.02 % (w/v) aqueous fast green for 3 minutes.
Rinse for approximately 10 seconds in 1% acetic acid.
Dip the slides in tap water briefly and then remove excess water from the slide.
Stain in 0.1 % (v/v) aqueous safranin-O for up to 5 minutes maximum.
Rinse in 100 % EtOH to remove extra red staining on the slide.
Dehydrate in 95% EtOH 2 × 5 minutes, followed by 100% alcohol 2 × 5 minutes.
Clear in xylene 3 × 2 minutes.
Mount with Permount.

Option E: Toluidine Blue

Re-hydrate in 100% EtOH 2 × 5 minutes, 95% EtOH 1 × 2 minutes, and 70% EtOH 1 × 2 minutes.
Wash in tap water for 30 sec.
Stain in 0.25 % (w/v in distilled water) aqueous toluidine blue for 5 minutes.
Rinse with distilled water until excess stain is washed away.
Dehydrate in 95% EtOH 2 × 5 minutes, followed by 100% alcohol 2 × 5 minutes.
Clear in xylene 3 × 2 minutes.
Mount with Permount.

Option F) Immunohistochemistry (IHC)

Deparaffinize by washing slides 3 times × 2 minutes/wash in xylene.
Dip slides in 100% EtOH for 2 washes of 2 minutes each
After slide dries, circle section with PAP pen
Rehydrate slides: 95% EtOH for 2 washes of 2 minutes each; 80% EtOH for 1 wash of 2 minutes; 50% EtOH for 1 wash of 2 minutes; D-PBS for 1 wash of 5 minutes
Quench endogenous peroxidase activity by submerging slides in 1 part 30% H₂O₂: 9 parts methanol for 10 minutes.
Wash slides in D-PBS: 3 washes of 2 minutes each
For antigen retrieval, add sufficient Digest-All to completely cover each tissue section
Incubate at RT for 5 minutes
Wash slides in D-PBS: 3 washes of 2 minutes each. (If not labeling for chondroitin sulfate, skip to stepXii)
If using antibodies for labeling chondroitin sulfate epitopes, apply chondroitinase ABC to each section and incubate at RT for 20 minutes.
Wash slides in D-PBS: 3 washes of 2 minutes each.
Block sections with Serum Blocking agent (Reagent A in kit)
Add enough reagent to completely cover tissue section (2–3 drops) TROUBLESHOOTING
Incubate at RT for 30 minutes (ensure sections do not dry)
Blot excess serum from bottom of inclined slide (do not rinse)
Dilute primary antibody in non-immune serum, Reagent A, or 10% goat serum (Col I, Col X – 1:400, C-4-S and C-6-S – 1:200, and Col 2 – 1:1).
Add antibody onto (+) staining sections, and blocking serum onto (−) control sections.
Incubate at room temperature for 1 hour or overnight at 4°C in large plastic petri dish lined with wet filter paper to keep slides moist.
Wash slides in D-PBS: 3 washes of 2 minutes each,
Apply secondary antibody (Reagent B in kit) to cover tissue sections (2 drops).
Incubate at RT for 10 minutes
Wash slides in D-PBS: 3 washes of 2 minutes each
Apply enough Enzyme Conjugate (Reagent C in kit) to cover tissue sections (2 drops)
Incubate at RT for 10 minutes
Wash slides in D-PBS: 3 washes of 2 minutes each
Add enough Substrate-Chromagen (AEC) mixture to cover tissue (2 drops)
Incubate at RT for 20 minutes
Gently dip sections in distilled H₂O (leave wet)
If desired, add 1 2 drops Hematoxylin to counterstain nuclei. If slides are not counterstained, skip to step XXXiii.
Incubate at RT for 5 minutes
Wash with tap H₂O
Wash slides in D-PBS for 30s or until slides turn a blue hue.
Wash slides in dH₂O: 3 washes for 2 minutes each (let slides remain in H₂O until coverslips are placed)
Mount slides by applying GVA Mounting Medium to 1 slide at a time and place coverslips.

CRITICAL: do not allow slides to dry before placing mounting medium.

Option G) Real-time quantitative RT-PCR (qPCR) methods

For gene expression analysis, first isolate the ASCs from the alginate matrix. Note that gene expression analysis can also be performed on pellets, but the RNA yield is much lower than using alginate beads. For experiments requiring gene expression for pellets, we suggest pooling 5–10 pellets and pulverizing the samples with a mortar and pestle cooled with liquid nitrogen before proceeding with the RNA isolation kit.
Add RNAsecure^™ reagent to the sodium chloride, sodium citrate buffer (final concentration, 1X) and distribute to 2 ml microcentrifuge tubes for each construct (3 beads) plus one for temperature control.
Heat solution to 60° C for 10 minutes on a hot plate to activate the RNAsecure^™ enzymes.
Cool the solution to 41° C, and add 1 construct (3 beads) to each tube.
Agitate tubes every 10 minutes until alginate is in solution. This should take ~ 1 hour. CRITICAL: maintain the solution at 41° C during this entire process to maintain enzyme activity.
Once alginate is no longer visible, centrifuge microcentrifuge tubes at 300 g for 5 minutes at 21° C to pellet cells.
Wash the cells with 500 ml of D-PBS with 1x RNAsecure and centrifuge again for 5 minutes at 21° C to pellet cells.
Aspirate D-PBS.
Either immediately process the cells with the RNA isolation kit or snap freeze in liquid nitrogen and store at −80° C for future processing. PAUSEPOINT Can be stored at −80° C for up to 1 month prior to further processing, though the user is cautioned that the samples should be processed as soon as possible to avoid any potential RNA degradation issues. 29. Assess gene expression by following the procedures of Nolan and Bustin ⁶⁷.

TIMING

Steps 1–19 ASC Isolation (3–5 hours for isolation)

Steps 20–28A ASC Expansion (2–5 weeks for expansion)

Step 28B Freeze Cells (2 Hours)

Step 28C ASC Pellet Culture (2 hours to prepare pellets)

Step 28D ASC Alginate Culture (4 hours to embed ASCs in alginate)

Steps 29A–B Biochemical determination methods (1 hour at appropriate time points to harvest samples and prepare for overnight papain digestion (Step 29). At a later time, dsDNA (Step 30, 2 hours), DMB (Option A, 1 hour), and OHP (Option B, ~10 hours over 2 days)

Steps 29C–F Histologic and immunohistochemical methods (Option C, 1 hour to fix samples overnight, 1 day to embed samples, 15 min/sample for sectioning, several days for staining). Safranin-O/Fast Green (Option D, 1.5 hours), Toluidine Blue (Option E, 1 hour), Immunohistochemistry (Option F, 24 hours).

Step 29G Real-time quantitative RT-PCR (qPCR) methods (2–3 hours at appropriate timepoints to isolate cells and 2 days for subsequent RNA isolation and qPCR at a later time).

ANTICIPATED RESULTS TROUBLESHOOTING

ASCs can be expanded rapidly in monolayer culture (Figure 2). Following expansion, ASCs can be differentiated in pellet or alginate culture accordingly, using the induction cocktails listed in Table 3. We have previously defined successful chondrogenesis using histologic and biochemical markers. For pellet culture, one can anticipate successful chondrogenesis as having sulfated glycosaminoglycan content greater than ≥ 931.7 ± 222.3 ng/pellet and having collagen II present in ≥ 29.0 ± 2.2% of the area stained per field of view ³⁷. This metric was based on histogram-derived thresholds set at values exceeding the 90th percentile of controls for each assay ³⁷. Similar metrics can also be applied to hydrogel based scaffold systems (e.g., alginate) to discern the degree and success of chondrogenesis; though the reader is reminded that due to the negative charge of the alginate matrix, neither safranin-o nor toluidine blue may be used. Therefore, when using alginate, the user of this protocol must rely on immunohistochemistry to discern the spatial organization of the cartilaginous matrix ^{45, 51, 56, 57}. Figure 4 demonstrates the variability observed in the accumulation of a sulfated glycosaminoglycan matrix in pellet culture using toluidine blue showing a positive result in 4b; whereas 4c is indicative of a poor or negative result for chondrogenesis. Similarly, under appropriate conditions, ASCs embedded in alginate synthesize collagens and proteoglycan (Figure 5); though negative markers of chondrogenesis (e.g., collagen I and excessive collagen X) should also be monitored. Typically, cartilage-specific matrix molecules are seen most intensely in the pericellular matrix with diffuse staining throughout the tissue-engineered construct. During chondrogenic differentiation, it is also typical to observe a significant size increase in the pellet. This pellet size difference is observed as early as 14 days in culture and persists throughout the duration of the experiment (Figure 6).

Figure 4

Toluidine Blue stain on (a) human articular cartilage and (b) and (c) are representative images of typical variation in GAG synthesis and accumulation obtained from ASC pellets cultured for 14 days. (Note. Expect similar results if Safranin-O stain were …

Figure 5

Representative immunohistochemistry results for chondroitin-4-sulfate and types I, II, and X collagen for a typical experiment with ASCs encapsulated in alginate after 4 weeks in in vitro culture. Positive Control: porcine cartilage for C-4-S, Collagen …

Figure 6

Pellet size after 6 weeks of culture. (a) representative image of pellets in 15 ml conical tubes in incomplete chondrogenic medium + 10% FBS on the left and complete chondrogenic medium containing TGF-b on the right and (b) the left two pellets were cultured …

In terms of biochemical content, typical standard curves for both the DMB and OH-proline assay are seen in Figure 7. In general, we measure S-GAG and OH-proline content in the 4 – 10 μg/μg of dsDNA as we have previously demonstrated ^34, 35. Our qPCR analyses have typically been used to ascertain early chondrogenic events. For example, after 7 days in culture, the addition of 500 ng/ml BMP-6 up-regulated the expression of principal cartilage ECM components, aggrecan (AGC1) and type II collagen (COL2A1) by an average of 205-fold and 38-fold, respectively over day-0 controls, while down-regulating the expression of type X collagen (COL10A1) expression by approximately 2-fold ⁴⁵; though it should be noted that qPCR data must be evaluated in concert with the other assays detailed in this protocol to determine chondrogenic efficiency. This is also true, in general, as the achievement of a specific value or threshold, in terms of biochemical content or fold-difference over control through qPCR analyses, does not necessarily indicate successful chondrogenesis. The degree and success of chondrogenic differentiation must ultimately be assessed by the investigator when viewed in terms of the hypothesis, objectives, the chondrogenic culture system, and the sum total of all assays employed to assess chondrogenesis.

Figure 7

Typical DMB (A) and OH-Proline (B) assay standard curves. n=3 wells per concentration. Data are mean values ± SEM.

Troubleshooting

See Troubleshooting guidance in table 6.

Table 8

Troubleshooting table.

Table 7

Hydroxyproline standards

Acknowledgments

The development of the protocols presented herein would not have been possible without the many contributions of Hani Awad, Geoffrey Erickson, Beverley Fermor, Yuan-Di Halvorsen, Kristen Lott, Henry Rice, Robert Storms, David Wang, Quinn Wickham, and Art Wu. This work was supported in part by an NSF Graduate Fellowship (BOD), the Coulter Foundation, the Duke Translational Research Institute, and NIH grants AR50245, AG15768, AR48182, AR48852.

Footnotes

AUTHOR CONTRIBUTIONS

BTE, BOD, JMG, and FG were involved in the development, testing, and troubleshooting of these protocols, as well as the writing of this manuscript. All authors contributed extensively to this work.

Competing interests statement:

The authors (BTE, JMG, and FG) have filed patent applications on some of the material contained in this article.

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Increase Height And Grow Taller Using Chondroitin And Glucosamine (Breakthrough?)

Just yesterday a reader of the website sent to me over the email link a very interesting video and a story from The Daily UK. On the video a fully grown man who is very bulked up from weight lifting talked about him increased his height in his usual sneakers from a little over 6′ 3″ to 6′ 4.25″, which he says would be a 1.25″ increase of height over a time span of 2.5 months. The Youtube video is from link HERE. The Account name is “ugoarimo” which the speaker shorten to just “ugo” from the beginning of the video. It seems that he had symptoms of knee pain resurface from high school days so he decided to take start taking the glucosamine sulfate and chondroitin.

Now, from a completely scientific point of view, I have seen at least 2-3 PubMed studies where patients with degrading thickness of articular cartilage in their knees resulting in knee pain had a reduction in knee pain after taking the glucosamine supplement for about 2-3 months. That could indicate that maybe, just maybe the glucosamine can actually no just halt the decrease in thickness of the articular cartilage, but also increase the thickness. However , that should still not explain why this man on the video claimed that his height increase by 1.25″ which is far beyond what I have thought glucosamine supplement could do.

So a little more about this guy who calls himself “ugo”. His other name that he goes by is The Black Spiderman. He seems to be some type of nutrition and fitness celebrity because on his Facebook page he has over 22600 likes. On his Twitter account he has over 17500 followers. He describes himself as a Physique Competitor/Model and BJJ Black Belt so he definitely tries to stay in shape. The video was published in July 2012 and he has almost 240 videos on his youtube account as of this time. On the other videos, he is not promoting any type of health supplement or multivitamin so if I was to say he was just another internet marketer trying to sell a product which is ineffective, I would have to second guess myself because I might be wrong.

The Science and Actual Study: From the article below…”The supplement is an amino acid made from combining sugar and glutamine. It is combined with sulphur to form glucosamine sulphate.” It seems that the key is to take the glucosamine sulphate. It is thought glucosamine improves water retention in the discs, which is likely to reduce wear and tear on the vertebrae. In the study, 36 people were asked to take the supplement for 4 weeks. Half of them took 1500mg of glucosamine sulphate daily. There was a control group given a placebo. The results seem to show that the group taking the Glucosamine Sulfate supplement at 1500 mg daily for about 1 month saw an increase in height of about 2-4 millimeters, which to me is very difficult to determine whether it could be from measurement error or real height increase. What is quite conclusive about the benefits of this supplement is it “halted the progression-of osteoarthritis in the knees of patients taking it daily for a few years“. If I was a betting man, I’d say that the height increase can be that the height decrease throughout the day was decreased and that some lost height was restored but the body in general never increased in height. However, I might be wrong. In the comments “Ugo” states that he started to experience bilateral knee pains just like in high school when he was still growing. [Note: They are referring to glucosamine sulfate, NOT glucosamine chondroitin.]

Additional Notes: Under the video where the comments are we see that a few people responded to him and said ….”Dude, i grew an inch. I accidentally bought the sulfate kind but i guess it still works. I’ve been taking like 3 2000mg pills a day (1 in the morning, 2 before bed) with meals for about 2 weeks. Basically ever since i saw your vid. I know it’s probably the cartilage in my spine regenerating, but idgaf. So thanks bro! I’m gonna keep taking this until i see no more changes“. So apparently another guy increased his height by 1 inch from taking the glucosamine sulfate at 2000 mg 3 times a day for only 2 weeks! Most of the other commenters called this guy a liar and said he didn’t grow but he seems to have a rather stoic and poker faced attitude to the accusations so judging him only by his actions and reactions to other people calling him a fraud, I’d say that I’m on the side that believes in what he claims, at least right now.

The thing is The Black Spiderman (as he calls himself) cites a link http://www.dailymail.co.uk/health/article-142809/Can-pill-make-taller-weeks.html which made me even more curious. Here is the entire article below…

Can a pill make you taller in four weeks?

by JENNY HOPE, Daily Mail

Can a pill really make you taller?

Glucosamine – a popular dietary supplement that seems to prevent the wear and tear of ageing joints – may also help people to grow taller.

New research suggests glucosamine sulphate, a naturally occurring building block for cartilage, can prevent shrinkage of the spine.

Top athletes have used glucosamine for years – and latest research shows the overthecounter supplement appears to protect cartilage tissue in the spine to the extent that it leads to an increase in height.

Glucosamine sulphate is believed to recondition cartilage tissue, rather than just counter the pain caused when joints naturally suffer osteoarthritis with increasing age.

Dr Peter McCarthy, from the Welsh Institute of Chiropractic at Glamorgan University, who carried out the study, said people taking glucosamine for four weeks grew taller.

It is thought glucosamine improves water retention in the discs, which is likely to reduce wear and tear on the vertebrae. As we age, we lose the ability to manufacture sufficient levels of glucosamine.

Dr McCarthy said: ‘It’s the first time research has shown that glucosamine, a nutritional supplement, can lead to an increase in height. We believe it has a direct effect on spinal joint tissues by preventing water loss from the cartilage in the discs.’

Glucosamine helps form new connective molecules that make vital links between cells and tissue, especially in cartilage.

The supplement is an amino acid made from combining sugar and glutamine. It is combined with sulphur to form glucosamine sulphate.

The new study involved 36 volunteers, half of whom took 1500mg of glucosamine sulphate daily. The remainder took a placebo pill manufactured to look the same.

After four weeks, two independent examiners found a slight increase in height among those taking the supplement.

There was no significant difference among those taking the placebo pill, says a report in the European Journal of Chiropractic 2002.

Dr McCarthy said: ‘The increase was not huge – it was between two and four millimetres – but this is a significant finding-Osteoarthritis affects an enormous number of people.

‘Glucosamine could be helping to reverse the pressures caused by standing upright. It could also be regulating the production of cartilage components. Supplemenation may either increase the total body height of the average person or reduce the amount of normal spinal shrinkage during the day.’

Thousands of people regularly use the supplement, which was introduced to Britain by David Wilkie, the former Olympic gold medal swimmer and managing director of Health Perception, which manufactures an oral supplement and a gel formula which can be rubbed into a painful joint.

Wilkie discovered glucosamine while training in the U.S. and has used it for more than 20 years.

Glucosamine is the preferred treatment for arthritis in Spain, Portugal and Italy.

Research in The Lancet medical journal last year found that the supplement halted the progression-of osteoarthritis in the knees of patients taking it daily for three years.

England rugby star Lawrence Dallaglio has taken glucosamine for years and believes it contributed to his comeback after injury. He said: ‘It works for me, but it’s good to see research which actually proves it’s having a beneficial effect.’

The Connection Between Aggrecan, Chondrogenesis, And Height

A compound or protein that I have been reading a lot about in the literature is aggrecan and it seems that have some direct influence and effect on chondrogenesis and the overall height. This is my attempt to do some basic research on Aggrecan.

I start off by just reading the Wikipedia article on aggrecan to get a general idea on what it is and what it does. The first few sentences makes me realize that aggrecan may be a not more important than I had previously believed.

“Aggrecan also known as cartilage-specific proteoglycan core protein (CSPCP) or chondroitin sulfate proteoglycan 1 is a protein that in humans is encoded by the ACAN gene. This gene is a member of the aggrecan/versican proteoglycan family. The encoded protein is an integral part of the extracellular matrix in cartilagenous tissue and it withstands compression in cartilage…Aggrecan is a proteoglycan, or a protein modified with large carbohydrates”

We remember that in at least the hyaline type cartilage found in the epiphyseal growth plates, the chondrocytes did excrete two main types of waste which would go on to make the extracellular matrix of the cartilage, type II collagen and proteoglycans. I had only briefly touched on looking at proteoglycans in the past in a very early post “The Effect On Height By Proteoglycans“. Looking back on these older posts I realize that I had no idea what I was doing or researching and understood very little of the details and mechanics of the cartilage that form the growth plates. I was trying and that is what I think is important.

From the name itself of aggrecan “cartilage specific proteoglycan core protein” it suggest that this type of protein is a type of proteoglycan which is either mainly or only found in cartilage. The fact that another name for it is chondroitin sulfate proteoglycan 1 shows that it may be similar to to the glycoaminoglycans we saw in the past or the non-sulfated type like the hyaluranon (aka hyaluronic acid)

From Wikipedia…

Aggrecan is a high molecular weight (1×10⁶ < M < 3×10⁶) proteoglycan. It exhibits a bottlebrush structure, in which chondroitin sulfate and keratan sulfate chains are attached to an extended protein core.

Aggrecan has a molecular mass >2,500 kDa. The core protein (210–250 kDa) has 100–150 glycosaminoglycan (GAG) chains attached to it. Along with type-II collagen, aggrecan forms a major structural component of cartilage, particularly articular cartilage.

What we are seeing is that aggrecans make up a huge major component in the matrix of most cartilage. When originally we were looking at the composition of cartilage, we only called them by the names, Collagen Type 2 and Proteoglycans. Now we can be more specific in seeing that one major type of proteoglycan found in at least articular cartilage is the aggrecan. The aggrecan itself seems to be much bigger than any glycoaminoglycan we saw before. There is a core protein part, and from the core protein projects about a couple hundred GAG chains, many of them being chondroitin sulfate and keratan sulfate.

If we remember from our past research on trying to find some type of supplement which can possible increase height, two major contenders were glucosamine sulfate and hyaluronic acid while another type, the chondroitin sulfate was very big as well. What we see from supplement stacks used from GrowTallForum.com was the use of glucosamine and chondroitin being used extensively. This suggest that GAGs have been the major source type of biological protein we were taking. At some point, after doing research on all three components, I tentatively decided that glucosamine sulfate and hyaluronic acid were two of three types of supplements which had the best chance of leading to some height increase for physically mature adults, even though those results might have been only a few milimeters of extra height at best.

It seems that overall, the aggrecan has 4 major areas, with 2 areas being on the N end and one area in the C end. There are areas in the compound called G1, G2, and G3 which have the function of chondrocyte apoptosis, hyaluronan binding, aggregation, and cell binding.

From the section for functions….

Aggrecan plays an important role in mediating chondrocyte-chondrocyte and chondrocyte-matrix interactions through its ability to bind hyaluronan

I am not sure about this but I would be willing to make a guess at this point that when the wikipedia article is referring to chondrocyte-chondrocyte interactions, the issue of orientation and chondrocyte arrangement might be included. One of the domains called G1 seems to bind to hyaluranon and proteins and form big complexes of protein that is 3-parts. This might involve a hyaluranon-aggrecan-protein system.

This next part in the wikipedia article was extremely englightening…

Aggrecan provides intervertebral disc and cartilage with the ability to resist compressive loads. The localized high concentrations of aggrecan provide the osmotic properties necessary for normal tissue function with the GAGs producing the swelling pressure that counters compressive loads on the tissue. This functional ability is dependent on a high GAG/aggrecan concentration being present in the tissue extracellular matrix.

This shows that there is a clear direct link between having extra aggrecan & GAGs and resisting the loss of height from intervertebral disk compression from gravitational loading. This is not only the first indication that taking certain GAGs may indeed lead to certain height increase like chondroitin sulfate and hyaluronic acid, but that any process to increase aggrecan especially in the cartilage that are part of a joint can lead to some height increase.

At the end of the clinical significance section it says…

Osteoarthritis is characterized by the slow progressive deterioration of articular cartilage. Cartilage contains up to 10% proteoglycan consisting of mainly the large aggregating chondroitin sulfate proteoglycan aggrecan.

It seems to validate the idea that the majority of proteoglycan types found in cartilage is the aggrecan. Since chondroitin sulfate does form a huge part of the chains that make the globular parts of the aggrecan, maybe taking chondroitin sulfate and keratan sulfate can really do lead to at least some decreased height loss from intervertebral decompression throughout a day.

Conclusion:

From only a quick analysis of just the Wikipedia article on aggrecan, I am willing to guess that this large complex of a protein is critical in giving the structure and strength of cartilage in general. I would say that aggrecan can lead to some height increase if one can get enough of hyaluranon and aggrecan in the right cartilage regions in one’s body. I am quite confident in showing that getting enough GAGs and aggrecan into one’s system can possibly lead to lead height loss throughout the day and may also help prevent the onset of osteoarthritis.

Increase Height And Grow Taller Using Chitosan

Another compound I have heard people claim could possibly increase height was over the compound Chitosan. When I first googled “chitosan grow taller” the first link was to the site GrowTall.com.

The website states that chitosan is a type of “fiber” that is made from crustacean shells from a chemical process. Besides being found in shrimp, lobsters, it seems that chitosan can also be found from teh plastic part of the squid we remove which can not be eaten. The first thing that comes to my head is to remember that a lot of high increase pills have a compound made from seashells as well based on calcium, usually calcium carbonate. From high school biology we remember that human digestive systems can’t digest fibers unlike most plant eating animals like cows which have 4 stomachs. The chitosan would go into the intestine area, bond with ingested fats, and come out when we go #2 in the bathroom.

The website suggest that chitosan might be useful for weight loss, can be used in wound treatment (ie closure) and that it should not be used by children and/or pregnant women due to growth retardation. This claim would mean that chitosan would actually make growing children shorter than they would actually be, which is the opposite of what we are looking for. As fror doasge, 3-6 grams of the compound can be taken each day with food. It seems chitosan has the ability to remove the body of certain minerals which again shows that it is a compound that is more likely to retard and stunt growth that promote it. The fact that it is stated that it can be used for woud healing may indicate it does have some angiogenic properties.

As for medicinal or therapeutic uses, some studies suggest it can possibly lower cholesterol as well as treat kidney failure. For the kidney failure, the chitosan is said to combine with an toxic compounds and this causes the toxins to be excreted out when the chitosan as a fiber is excreted. From a very syperficial level, that is a reasonable scientific guess as why researchers may think it helps with kidney failure.

As for the claim that it is used to treat wounds and help in wound healing/closure, the idea is that along with the plasma, the chitosan may be able to bind to whatever usually like blood and plasma that comes along to close wounds and help built new tissue. That other idea is that chitosan may be able to kill some bacteria (like Strep) and yeast (like Candida)

Some studies claim it helps prevent or treat tumors, which would suggest that it is more catabolic in nature which is against what most height increase products would claim.

From the website GrowTall.com…

“Animal studies suggest that some forms of chitosan may help to prevent bone loss;… however, because chitosan also interferes with mineral absorption, the net effect in humans might actually be to increase bone loss…

Safety Issues

There is significant evidence that long-term, high-dose chitosan supplementation can result in malabsorption of some crucial vitamins and minerals including calcium, magnesium, selenium, and vitamins A, D, E, and K.33,34 In turn, this appears to lead to a risk of osteoporosis in adults and growth retardation in children. For this reason, adults taking chitosan should also take supplemental vitamins and minerals, making especially sure to get enough vitamin D, calcium, and magnesium.”

The overall conclusion on this resource is that chitosan is more like to remove important minerals for bone instead od prevent bone loss. If this is the case, for children who are still growth, that can indeed interfere with with the endochondral ossification process. Let’s not forget that while a lot of our research now is on cartilage and cartilage regeneration, for us to have the height we already do, it does involve the fact that calcium and otehr hard deposits have been layered and grown on top of each from the natural height growing process. Bone and mineral loss is a problem for people still growing.

However, the effects on people who are finished done growing who wish to do exercises to lengthen long bones may be the exact opposite. I do note that there has been some weak positive correlation with the idea that weaker bones with lower BMD (bone mass density) may mean that they are easier to change and be melleable (if only a little) to some bone modeling loading. If chitosan can remove the minerals in the bones that make them so hard, it might indeed assist in some techniques we have talked before like the shinbone method, in inducing microfractures, and the LSJL method.

As for the conclusion of this source on Chitosan, I say that there is a slight chance that Chitodan may help people who are already finished with the natural growth process grow taller if they take it long enough and but also did some type of bone remodeling exercises.

A post on the prospective affects by Chitosan was looked at already by HeightQuest.com in “Height increase with Chitosan?” .

Analysis:

His main point is to get the chitosan to actually go into the blood stream for some effect instead of getting it completely passed through the digestive tract without it ever getting used. The difficulty is to get the chitosan content into the bone marrow. From one study he quotes “Effect of dietary supplementation of chitosan and galacto-mannan-oligosaccharide on serum parameters and the insulin-like growth factor-I mRNA expression in early-weaned piglets.” Chitosan might help because it will “encouraging epiphyseal bone marrow stem cells to become a more rounded pro chondrogenic shape”

The last resource I would raise is another result found from Google on a forum thread from Soompi HERE. A member named Jaeho would write…

“Also, my mom ordered Royal Jelly and something called SmarTall. SmarTall is apparently the kids’ version of Royal Jelly. My brother and sister have been taking SmarTall for 2 days.

I also searched Chitosan online, but all the sites say it’s a weight loss pill and that it’s a scam… BUT in Korea, it’s marketed as a miracle product that cures all sorts of health problems. I can’t find anything on SmarTall though.”

Anyway, I asked my mom why she bought SmarTall and she said it was like Royal Jelly for children… but it’s for height too? Well, the name gives it away… lol. From a first guess”

Analysis:

It would seem that chitosan is being marketed in east asian countries like Korea for being a sort of miracle pill that has multiple therapeutic benefits. It’s claims to help people loss weight makes it very attractive to the Korean people who are well known by being very appearance conscious. There is no claim that chitosan has any effect on height but there is another product that is Royal Jelly derived called SmarTall being sold (at least back in 2006) in Korea which is given to kids to help them grow taller. The conclusion here is that Chitosan has no height increasing properties.

If we type in the term “chitosan height increase’ into google instead what we find are studies which do suggest that chitosan does have some height and size increasing ability but those are just for vegetables, specifically grains.

A link to a webpage on Vanderbilt University HERE shows that people have already looked into chitosan quite extensively and summarized its properties and possible effects. They conclude with….

“Although companies selling chitosan claim that the product is very effective and claim to have medical research about how well their product works, the medical studies show that when taken alone, chitosan has not been proven to help a person lose weight, increase their HDL cholesterol, or decrease their LDL cholesterol. Instead, chitosan can cause unwanted gastrointestinal cramps and constipation”

Conclusion:

Chitosan may be harmful to growing children in terms of growth retardation from its ability to absorb important minerals the body and bones need when the person is still growing. After the person is finished growing, there might be a small chance that a person can use chitosan’s mineral absorbing properties to make bones less hard and easier to model so it might be possible to use it with some bone loading technique to lengthen bone.

Using BMP-6 To Differentiate Adipose Derived Adult Stem Cells Into Chondrocytes And Cartilage Regeneration

Me: This post is to show that another option of growth factors we can use to turn the adipose derived adult stem cells into chondorcytes is to use BMP-6. Remember that in the long bones of adults, the marrow is considered yellow and mostly made of fatty acids. The study showed that TGF-Beta1 has also been shown to work, causing the expresion of cartilage specific genes an proteins like aggrecan and type II collagen. There was actually 5 growth factors all looked at in this study. They are looking at the chondorgenic potential of these on ADAS cells in alginate beads

1. TGF-Beta 1
2. TGF-Beta 3
3. IGF-1
4. BMP-6
5. Dexamethasone

The two main points from the study are.

1. BMP-6 up-regulated AGC1 and COL2A1 expression by an average of 205-fold and 38-fold, respectively, over day-0 controls, while down-regulating COL10A1 expression by approximately 2-fold.

2. BMP-6 is a potent inducer of chondrogenesis in ADAS cells, in contrast to mesenchymal stem cells, which exhibit increased expression of type X collagen and a hypertrophic phenotype in response to BMP-6.

So for future reference, we realize that if we wanted to inject the BMP-6, we would put it in the bone marrow part, not the epiphysis part since the MSCSs only caused COL10 expression and hypertrophic phenotype. Any height increase method we create will have growth factors added in certain areas in certain combinations in certain sequences,

From PubMed study link HERE

Arthritis Rheum. 2006 Apr;54(4):1222-32.

Potent induction of chondrocytic differentiation of human adipose-derived adult stem cells by bone morphogenetic protein 6.

Estes BT, Wu AW, Guilak F.

Source

Department of Surgery, Duke University Medical Center, Durham, North Carolina 27710, USA.

Abstract

OBJECTIVE:

Recent studies have identified an abundant source of multipotent progenitor cells in subcutaneous human adipose tissue, termed human adipose-derived adult stem cells (ADAS cells). In response to specific media formulations, including transforming growth factor beta1 (TGFbeta1), these cells exhibit significant ability to differentiate into a chondrocyte-like phenotype, expressing cartilage-specific genes and proteins such as aggrecan and type II collagen. However, the influence of other growth factors on the chondrogenic differentiation of ADAS cells is not fully understood. This study was undertaken to investigate the effects of TGFbeta1, TGFbeta3, insulin-like growth factor 1, bone morphogenetic protein 6 (BMP-6), and dexamethasone, in various combinations, on the chondrogenic potential of ADAS cells in alginate beads.

METHODS:

The chondrogenic response of alginate-encapsulated ADAS cells was measured by quantitative polymerase chain reaction, 3H-proline and 35S-sulfate incorporation, and immunolabeling for specific extracellular matrix components.

RESULTS:

Significant differences in chondrogenesis were observed under the different culture conditions for all outcomes measured. Most notably, BMP-6 up-regulated AGC1 and COL2A1 expression by an average of 205-fold and 38-fold, respectively, over day-0 controls, while down-regulating COL10A1 expression by approximately 2-fold.

CONCLUSION:

These findings suggest that BMP-6 is a potent inducer of chondrogenesis in ADAS cells, in contrast to mesenchymal stem cells, which exhibit increased expression of type X collagen and a hypertrophic phenotype in response to BMP-6. Combinations of growth factors containing BMP-6 may provide a novel means of regulating the differentiation of ADAS cells for applications in the tissue-engineered repair or regeneration of articular cartilage.

PMID: 16572454 [PubMed – indexed for MEDLINE] Free full text

Using Microspheres With TGF-Beta1 And Chitosan To Differentiate Adipose Derived Stem Cells Into Chondrocytes And Repair Cartilage Defects

Me: This seems to suggest that we can create these hybrid microspheres which are just basically encapsulations or coatings of some collagenous fibrous material with TGF-Beta1 and/or Chitosan put inside and put it into the intermedullary cavity to get what little stem cells inside to differentiate into chondrocytes since the adult bone marrow is not red but yellow which is fatty acids and anything that is derives from fatty acids.

I have written in another article over the ability of BMP-6 to turn adiposed derived adult stem cells into chondrogenic in phenotype unlike for mesenchymal stem cells. It seems that the winner in this study was the hybrid microsphere with TGF-beta 1 and chitosan inside. This helps me further in deriving a height increase method. I know now that we can create microspheres for injection close to the bone marrow. Personally I would use the TFG-Beta 1 with Chitosan first which has a lesser chondrogenic effect and then added the BMP-6 encapsulations for further chondrogenesis.

From PubMed study link HERE

Joint Bone Spine. 2010 Jan;77(1):27-31. Epub 2009 Dec 22.

Cartilage regeneration using adipose-derived stem cells and the controlled-released hybrid microspheres.

Han Y, Wei Y, Wang S, Song Y.

Source

Department of orthopaedics, Xijing Hospital, The Fourth Military Medical University, West Road Changle, Xi’an, China. yishenghan@homtail.com

Abstract

OBJECTIVE:

This study was to evaluate the effect of hybrid microspheres (MS) composed of gelatin transforming growth factor-beta (TGF-beta1)-loaded MS and chitosan MS on the enhancement of differentiation of adipose-derived stem cells (ASCs) into chondrocytes in pellet culture in vitro and the reparative capacity of pellet from ASCs and the hybrid MS-TGF used to repair cartilage defects in vivo.

METHODS:

The morphology of the controlled-released MS was observed with scanning electron microscopy (SEM) and mechanical property was also tested in this study. In vitro TGF-beta1 release was evaluated by an enzyme-linked immunosorbent assay. The protein expression of Collagen II was tested by Western blot. In addition, a preliminary study on cartilage regeneration was also performed in vivo.

RESULTS:

When chondrogenic differentiation of ASCs in both MS was evaluated, the protein expression of Collagen II became significantly increased for the hybrid MS-TGF, as compared with the gelatin MS-TGF. Mechanical result showed that the hybrid MS was superior to the gelatin MS. Observation of histology in vivo demonstrated that the pellet from ASCs and the hybrid MS-TGF promoted cartilage regeneration in the defects of articular cartilage much better than other groups.

CONCLUSION:

Our study demonstrated that the pellet from ASCs and the hybrid MS-TGF can provide an easy and effective way to construct the tissue engineered cartilage in vitro and in vivo.

PMID: 20022784 [PubMed – indexed for MEDLINE]