Bioengineered Cartilage Pellets And LIPUS For Longtitudinal Growth (Huge Breakthrough!)

Me: This is one of the only studies I’ve found so far which seems to be talking about the exact same thing we at this website have bene talking for the longest time. I know that this article has been looked at by Tyler on HeightQuest.Com HERE and I have looked at his analysis and critique very thoroughly since this study is as close to a real study on height increase as I have found so far. It has also been looked at on other height increase forums. I can’t find the complete article but Tyler seemed to have more than me.

This article is fascinating since it would appear that the rabbits actually had their leg bones fractured, then had both a bioengineering cartilage pellet, and the LIPUS technology administered.

Huge Breakthrough 1: The study states conclusively that if we decided to use a height increase method which involved distraction, using the LIPUS technology doesn’t help. Very useful tip here. Tyler argues over the mechanics about whether they might have administered it wrong but let’s assume these Ph. Ds knew what they were doing for the time being.

Huge Breakthrough 2: The study showed that a bioengineering chondrocyte pellet was implanted in an induced distraction/fracture area. This means that somehow these group of researchers from Hong Kong have finally actually managed to completely grow a growth plate cartilage in vitro which works. The reasoning I wanted to use is this. I thought from the previous post that these researchers could only develop hyaline cartilage but this study showed that they hav also been successful in making growth plate cartilage. if you tried implanting just ordinary hyaline cartilage in growth plate physis fracture you would not get the same type of longitudinal growth because their is no perichondrium with the blood vessels going through it. You need to have a functional growth plate to allow for that type of increase in longitudinal growth, which some thing like an articular cartilage extract would not be able to do.

Implications: This shows the first real non limb-lengthening procedure for bone lengthening ever. I know that some people might want to disagree since this is just an implant into the physeal (growth plate) injury but I am extremely confident that what the researchers have done is create a synthetic, immunologically non-resistant growth plate which can be implanted back into bone and grow.

[I would be willing to guess that the growing process they use to do the growth pellet formation was from this study HERE, which Tyler also cited in the same article post from february of 2012 which I linked above. And I have the complete study article on my post. ]

From PubMed study link HERE

J Biomed Mater Res B Appl Biomater. 2011 Oct;99(1):36-44. doi: 10.1002/jbm.b.31869. Epub 2011 Jun 16.

Restoration of longitudinal growth by bioengineered cartilage pellet in physeal injury is not affected by low intensity pulsed ultrasound.

Chow SK, Lee KM, Qin L, Leung KS, Cheung WH.

Source

Department of Orthopaedics and Traumatology, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong.

Abstract

Physeal fracture is a common pediatric fracture that would result in premature physeal closure in long bones, and there is currently no gold standard for its management. In this study, we investigated the application of a Bioengineered Cartilage Pellet (BCP) in repairing a rabbit physeal fracture model, and the possible effects of Low Intensity Pulsed Ultrasound (LIPUS) treatment. Rabbits with physeal fracture created were assigned to the NC group (no BCP, no LIPUS), GC group (BCP, no LIPUS), and GT group (BCP and LIPUS). Femoral lengths and cartilage area were assessed at 4, 8, and 16 weeks post-defect. After transplantation, the BCP showed continuous growth in the host and demonstrated resemblance to a natural growth plate. The GC group showed 34.1, 32.1, and 41.1% advantage in lengthening over the NC group and the GT group showed 51.1, 41.6, and 26.9% improved lengthening than the NC group, at 4 (p = 0.203), 8 (p = 0.543) and 16 weeks (p = 0.049), respectively. Cartilage area was shown to be significantly higher in GC and GT group compared to NC group (p < 0.05). No significant difference was found between GC and GT group. Femoral longitudinal growth was shown to be improved by the BCP, however no additional enhancement effect was shown to be provided by LIPUS.

PMID: 21681954 [PubMed – indexed for MEDLINE]

Hyaline Cartilage Engineered By Chondrocytes In Pellet Culture Compared To Cartilage Implants (Important)

Me: This is one of those studies which will be critical if we ever choose to do a more invasive method to increase height possibly through using implants from in vitro cultured chondrocyte pellets. I have always been slightly worried over the idea of whether the cartilage the researchers can grow and develop in the cell dish can be as similar to the native cartilage and this study has given me definite proof that from using condensation of chondrocytes in cultured pellet, with the right mediums can create the real thing. This is one huge step towards showing that regrowing an entire new growth plate is possible. However it is important to remember that although a hyaline cartilage was regrown to almost perfect in vivo statues, it is not growth plate hyaline cartilage. There is still a big difference.

Here is what I always suggest the reader do: Read the 1. abstract, 2. introduction, 3. discussion, and 4. conclusion!

Analysis: It seems that if you extract some cartilage and try to regrow the chondrocyte in the extraction, if it is a monolayer it turns into a type of fibroblastic morphology. It starts releasing Collagen type I, instead of type II and it’s aggrecan and collagen type II core protein disappears. The process of chondrocyte condensation can actually reverse the differentiation process when in some type of cell medium. This seems to be due to the cell density being decreased in the monolayer. If you put the chondrocytes in a packed 3-D form of pellet, the chondrocyte cell density is kept high which keeps it from reversing into the differentiation process and condensing.

From the study the researchers stated that for the grown culture to be the same as the native culture, they have to be structurally and biochemically the same. The grown material was tested immunohistochemically using the different types of collegan and aggrecan. From the discusssions section “After 1–2 weeks in culture, the neocartilage shared similarities with native cartilage with regard to chondrocyte phenotype, matrix distribution and the ultrastructure of collagen fibrils.”

The last sentence of the study abstract is what made me realize that we are one more step closer to a real height increase solution…”In conclusion, hyaline cartilage engineered by chondrocytes in pellet culture, without the transformation of cell phenotypes and scaffold materials, shares similarities with native cartilage in cellular distribution, matrix composition and density, and ultrastructure.”

Note: Tyler, you said in February 2012 where you analyzed this same study that you didn’t have access to the entire study but you can find it now here on this website.

From PubMed study link HERE…

J Anat. 2004 September; 205(3): 229–237.

doi: 10.1111/j.0021-8782.2004.00327.x

PMCID: PMC1571343

Hyaline cartilage engineered by chondrocytes in pellet culture: histological, immunohistochemical and ultrastructural analysis in comparison with cartilage explants

Zijun Zhang,¹ J Michael McCaffery,² Richard G S Spencer,¹ and Clair A Francomano¹

Abstract

Cartilage engineering is a strategic experimental goal for the treatment of multiple joint diseases. Based on the process of embryonic chondrogenesis, we hypothesized that cartilage could be engineered by condensing chondrocytes in pellet culture and, in the present study, examined the quality of regenerated cartilage in direct comparison with native cartilage. Chondrocytes isolated from the sterna of chick embryos were cultured in pellets (4 × 10⁶ cells per pellet) for 2 weeks. Cartilage explants from the same source were cultured as controls. After 2 weeks, the regenerated cartilage from pellet culture had a disc shape and was on average 9 mm at the longest diameter. The chondrocyte phenotype was stabilized in pellet culture as shown by the synthesis of type II collagen and aggrecan, which was the same intensity as in the explant after 7 days in culture. During culture, chondrocytes also continuously synthesized type IX collagen. Type X collagen was negatively stained in both pellets and explants. Except for fibril orientation, collagen fibril diameter and density in the engineered cartilage were comparable with the native cartilage. In conclusion, hyaline cartilage engineered by chondrocytes in pellet culture, without the transformation of cell phenotypes and scaffold materials, shares similarities with native cartilage in cellular distribution, matrix composition and density, and ultrastructure.

Introduction

The lack of self-healing capacity in cartilage and the considerable morbidity caused by cartilage injuries and diseases have encouraged the search for a biomedical solution for repairing or restoration of damaged articular cartilage. The recent progress in tissue engineering of cartilage includes the introduction of new biomaterials for the scaffold, development of a bioreactor culture system, and the investigation of various cell resources and use of growth factors (Cao et al. 1998; Vunjak-Novakovic et al. 1999; Potter et al. 2000; Temenoff & Mikos, 2000; Solchaga et al. 2001; Cancedda et al. 2003). However, at the present time engineered cartilage still does not satisfy the need for functional cartilage repair (Anderer & Libera, 2002).

The phenotypic instability of chondrocytes in conventional monolayer culture has been a great challenge to cartilage engineering. In monolayer culture, chondrocytes typically devolve to a fibroblastic morphology and secrete type I collagen into the matrix but lose the expression of type II collagen and aggrecan core protein (Schnabel et al. 2002). The dedifferentiated chondrocytes reverse their phenotype when condensed by continuous culture after reaching confluence (Schulze-Tanzil et al. 2002). However, it is not clear to what extent the original chondrocyte phenotype is recapitulated during the redifferentiation process (Schnabel et al. 2002). In order to produce functional cartilage, it is crucial to avoid chondrocyte dedifferentiation during the process of cartilage engineering.

Cell density is one of the critical requirements for stabilizing the chondrocyte phenotype. When chondrocytes are plated at a high density, e.g. 4 × 10⁵ cm⁻² in culture flasks, their phenotypes do not change (Ruggiero et al. 1993). Similarly, chondrocyte pellet culture has provided an in vitro model of cartilage mineralization, usually involving growth plate chondrocytes (Kato et al. 1988; Farquharson & Whitehead, 1995). There is a very great distinction in the fate of chondrocytes between the growth plate, which is progressing towards calcification, and hyaline cartilage, such as articular cartilage, which normally does not calcify (Pacifici et al. 2000). Although some studies have explored the use of pellet culture for growth plate chondrocytes and the process of mineralization, few studies have examined hyaline cartilage chondrocytes in pellet culture. Published papers using pellet culture for hyaline cartilage chondrocytes have studied the effects of exogenous agents such as bone morphogenetic protein on the chondrocyte phenotype (Stewart et al. 2000), properties of extracellular matrix (Larson et al. 2002) and bioenergetics of chondrocytes (Croucher et al. 2000; Graff et al. 2000). Presently, no previous work has used this method to engineer hyaline cartilage, and subsequently evaluated the characteristics of neocartilage. The current study, utilizing chondrocytes from a hyaline cartilage source, was designed to engineer cartilage by pellet culture. The rationale behind this method is: (1) that chondrocyte phenotype is stabilized in the pellet, thereby avoiding dedifferentiation–redifferentiation; and (2) that cartilage is engineered without additional materials such as supporting scaffolds or gels (Vunjak-Novakovic et al. 1999; Temenoff & Mikos, 2000), which introduce possible complications including immune/inflammatory responses (Cancedda et al. 2003).

The chick sternum has been widely used in chondrocyte and cartilage studies because of its easy access and unique characteristics of development (Gibson & Flint, 1985; Hirsch & Svoboda, 1998; Liu et al. 1999;Tew et al. 2000; Zhang et al. 2002). The proximal sternum calcifies between gestational days 16 and 17, preceded by chondrocyte hypertrophy. In contrast, the distal sternum maintains a hyaline phenotype and does not calcify during development (Gibson & Flint, 1985; Craig et al. 1987). Thus, only the distal part of the chick sternum was utilized in the present study. In order to be functional, the regenerated cartilage is required to be structurally and biochemically identical to native cartilage (Caplan et al. 1997). Native cartilage explants served as control in the present study. In this study, the chondrocyte phenotype was assessed with immunohistochemistry of types I, II, IX, X collagen and aggrecan. Matrix deposition and the formation of a fibril network at the ultrastrucutural level were compared between the engineered cartilage and cartilage explants taken from the same source.

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Materials and methods

Pellet and explant cultures

Fertilized White Leghorn chicken eggs (Truslow Farms, Inc., Chestertown, MD, USA) were incubated at 37 °C for 16 days. The distal part of the sternum was removed from chick embryos, predigested in 0.2% collagenase (Worthington Biochemical Corp., Lakewood, NJ, USA) for 30 min, and further digested in fresh collagenase solution for 3 h. Chondrocytes were suspended in supplemented Dulbecco’s modified Eagle medium (DMEM; Biofluids Inc., Rockville, MD, USA) at a concentration of 10⁷ mL⁻¹. Four × 10⁶chondrocytes in 0.4 mL of cell suspension were transferred into each 0.75-mL tube (Matrix Technologies Corp., Lowell, MA, USA). Chondrocyte pellets were formed by centrifugation at 500 g for 10 min. The culture medium was DMEM plus 10% fetal bovine serum (Hyclone, Logan, UT, USA), 50 µg mL⁻¹ascorbate (Sigma, St Louis, MO, USA), 2 mm glutamine (Biofluids Inc.) and 0.2% penicillin/streptomycin (Life Technologies, Rockville, MD, USA). The pellets were cultured at 37 °C under a gas mixture of 95% air/5% CO₂. The pellets were transferred into Petri dishes at day 3 and continued to grow for 2 weeks. The medium was changed every day for the first 3 days and every second day for the rest of the culture period. Cell viability, regularly monitored with the LIVE/DEAD® kit (Molecular Probes, Eugene, OR, USA), was greater than 90% throughout the culture period.

For explant cultures, the distal parts of sterna were diced up to about 1 × 1 × 1 mm in size. One piece of the cartilage in 0.4 mL DMEM was added into the 0.75-mL tube and centrifuged in parallel to the chondrocytes at the time of pelletting. The explants were cultured in the tubes before being moved into Petri dishes at day 3. The culture conditions and medium were identical to pellet culture.

Samples were taken at days 1, 3, 7 and 14 and fixed in 4% paraformaldehyde for 1 h. They were then washed in phosphate-buffered saline (PBS) and passed through series of sucrose gradients at concentrations of 10%, 25% and 50%, and finally embedded in OCT compound (Sakura Finetechnical Co. Ltd, Tokyo, Japan) and sectioned at 5 µm with a cryostat for histology study. Samples for electron microscopy were collected on the same time schedule as histology but fixed separately (see below).

Immunohistochemistry

The primary antibodies for immunohistochemistry were mouse anti-avian collagens type I (SP1.D8), II (II-II6B3), IX (2C2) and X (X-AC9), and aggrecan link protein (9/30/8-A-4), obtained from the Developmental Studies Hybridoma Bank, The University of Iowa (Iowa City, IA, USA). Sections were predigested with 2 mg mL⁻¹ hyaluronidase (Sigma) for 30 min, or 1 h for type IX collagen staining (Vilamitjana et al. 1989), for the optimal penetration of antibodies. Before applying 9/30/8-A-4, aggrecan was reduced and alkylated by incubation with dithiothreitol and iodoacetic acid. After blocking with normal rabbit serum, primary antibody diluted in 1% bovine serum albumin (SP1.D8 1 : 10; II-II6B3, 2C2, X-AC9 and 9/30/8-A-4 1 : 5) was added onto the sections and incubated overnight at 4 °C. The incubation for Cy3-conjugated secondary antibody (Biomeda Biotechology, Foster City, CA, USA) was 30 min at room temperature. Sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA). Primary antibody was replaced with PBS in the staining controls. Slides were viewed under an epifluorescent microscope and images were captured with a digital camera (Olympus BX51, Olympus America, Melville, NY, USA).

Histomorphometry

Histomorphometric analysis of aggrecan staining was performed on ten randomly selected sections of pellets and explants taken at scheduled time points, using Meta Imaging Series 4.6 (Universal Imaging Corp., West Chester, PA, USA). On each section, the measured areas, a computer-defined area of 350 × 350 pixels, were allocated as shown in Fig. 1. The average staining intensity in grey scale and the number of cells in the selected areas were calculated.

Fig. 1

Diagram of histomorphometry. On the pellet section, line c divides the section into two parts. Line b is the middle line of each part. The measuring area is on the medial side of line b. On the explant section, lines a and b are the intersect lines on …

Electron microscopy

Cartilage samples were processed as previously described (McCaffery & Farquhar, 1995). Briefly, samples for electron microscopy were fixed in fixative with 3.0% formaldehyde, 1.5% glutaraldehyde in 0.1 msodium cacodylate and 2.5% sucrose (pH 7.4) for 1 h at room temperature. They were then washed three times in 0.1 m sodium cacodylate/2.5% sucrose and post-fixed at 4 °C in Palade’s fixative containing 1% OsO₄. The samples were then washed, stained with uranyl acetate, dehydrated through a graded series of ethanol, and embedded in Epon. Eighty-nanometre sections were cut on a LEICA UCT ultramicrotome, post-stained in lead citrate and 2% uranyl acetate, and analysed on a Philips 420 TEM electron microscope (Royal Philips Electronics, The Netherlands) operated at 80 kV. The images were recorded and analysed with a Soft Imaging System Megaview III digital camera/software (Soft Imaging System Corp., Lakewood, CO, USA). Measurements of fibril diameter and the percentage of fibres were determined in 15 randomly selected views for the 2-week samples.

Statistical analysis

Data are presented as mean ± standard deviation and evaluated with Student’s t-test to compare the pellets with the explants at the same time point. Significance was defined as P < 0.05>

Results

The initial chondrocyte pellets, about 3.0 mm in diameter, at the bottom of the 0.75-mL tube became solid at day 3 when they were transferred from culture tubes into Petri dishes. The disc-like regenerated cartilage was about 9.0 mm in diameter and 1.0 mm in thickness by 2 weeks of culture (Fig. 2). When handling the samples for processing, the neocartilage showed similar rigidity and compression with the explant cartilage. The explants also grew and increased in size remarkably during the period of culture.

Fig. 2

Cartilage tissue generated by chondrocytes in pellet culture for 14 days. The initial number of chondrocytes was 4 × 10⁶ per pellet. The disc-shape tissue was hyaline in appearance (scale bar = 10 mm).

Immunohistochemistry and histomorphometry

Chondrocytes in both pellets and explants were round through the culture period. At day 1, chondrocyte density in the pellets (110 ± 17.9) was more than twice that in the explants (51 ± 9.5; P < 0.001). At day 3, a notable change in the pellets was the expansion of the intercellular space, accompanied by a gradual decrease in cell density in the pellets (80 ± 5). At day 7, cell density in the pellets (74 ± 8.5) was largely the same as in the explants (66 ± 8.4; P > 0.05), owing to the decreasing trend in the pellets and a peak increase in the explants. By day 14 cell density in the pellets (42 ± 7.8) and explants (37 ± 5.5; P > 0.05) was virtually identical.

Immunohistochemistry for aggrecan and types II and IX collagen demonstrated dynamic changes in matrix composition in both pellets and explants. Aggrecan was not seen around chondrocytes in the pellets until day 3 (Fig. 3). At day 3, the extracellular matrix in the pellets stained positively for aggrecan. Aggrecan in the pellets was mainly pericellularly stained, whereas it was uniformly stained throughout the extracellular space in the explants. The average intensity of aggrecan staining in the pellets was less than half that in the explants (Fig. 4). The average intensity of aggrecan staining in the explants steadily increased during the culture period. By day 7, aggrecan staining in the pellets showed the same pattern as in the explants and the average intensity in the pellets was equivalent to that in the explants (P > 0.05). At day 14, pericellular staining of aggrecan was the dominant pattern in both pellets and explants, and the average intensity was nearly identical.

Fig. 3

Immunohistochemistry for aggrecan on pellets (left column) and explants (right column). Reading from top to bottom, the rows reflect results at days 1, 3, 7 and 14. There was no aggrecan staining in the pellets, but was positively stained in the explants …

Fig. 4

The average staining intensity of aggrecan in the pellets and explants. Between days 1 and 3, aggrecan intensity in the pellets was significantly less then that in the explants. However, it was above the level of explants (P > 0.05) at day 7 and …

At day 1, type II collagen was not stained around chondrocytes in the pellets, whereas the extracellular matrix in explants was uniformly positive for type II collagen staining (Fig. 5). In the pellets, type II collagen staining was seen in the extracellular space at day 3 and was consistently positive up to day 14. In comparison with the explants, type II collagen was not evenly stained at day 7, and was more pericellularly distributed at day 14.

Fig. 5

Immunohistochemistry for type II collagen on pellets (left column) and explants (right column). Reading from top to bottom, the rows reflect results at days 1, 3, 7 and 14. Type II collagen was not stained in the pellets until day 3. At days 7 and 14, …

At day 1, type IX collagen was identified in the pellets and in the explants, and showed a more diffusive staining in the extracellular matrix of the pellets as compared with the explants (Fig. 6). The staining pattern was reminiscent of type IX collagen in both pellets and explants by day 3. Type IX collagen stained more intensely in the pellets in general, although it was unevenly stained in the matrix in both explants and pellets. By day 14, staining for type IX collagen in the pellets and explants shared the same pattern of both intracellular and extracellular staining.

Fig. 6

Immunohistochemistry for types IX (a–h) and X collagen (i, j) on pellets (left column) and explants (right column). Reading from top to bottom, the rows reflect results at days 1, 3, 7 and 14. Type IX collagen was positive in both pellets and …

Type I collagen was only stained at the edge of some of the pellet and explant sections. Type X collagen was negative in both the pellet and explant, even at the end of culture (Fig. 6I,j).

Ultrastructure

At day 1, chondrocytes in the pellets were at a high concentration, and some were next to each other. However, most of the intercellular space was electron-translucent (Fig. 7a). Chondrocytes in the explants were surrounded by highly organized matrix fibrils (Fig. 7b). Chondrocytes in both pellets and explants were rounded in shape, containing conspicuous nuclei with prominent endoplasmic reticulum and Golgi apparatus indicative of healthy, actively growing cells. At day 3, deposition of granular and fibril matrix was observed in the vicinity of chondrocytes in the pellets. The fibrils and macromolecules were sparse and randomly orientated around chondrocytes. No fibril network such as in the explants was formed (Fig. 7c,d). At day 7, the number of collagen fibrils was significantly increased in the pellets and the fibrils were assembled into a network. However, the fibril meshwork was still rather loose compared with that in the explants (Fig. 7e,f). Rough endoplasmic reticulum of chondrocytes in the pellets was less prominent than at day 3. Chondrocytes in the explants, but not in the pellets, showed an increasing number of vacuoles in cytoplasm. At day 14, the average density of collagen fibrils was 0.197 ± 0.066 in the pellets and 0.253 ± 0.054 in the explants (P = 0.103). The average diameter of collagen fibrils was not significantly different (pellets: 22.11 ± 3.32 nm; explants: 23.93 ± 2.89 nm, P = 0.085). However, there was a striking difference between the pellets and explants in the manner of fibril organization and orientation (Fig. 7g,h). Whereas collagen fibrils in the explants knitted into a well-connected network, the fibrils in the pellets appeared disorganized and randomly orientated.

Fig. 7

Pellets and explants: electron microscopy. At day 1, diminished electron density was seen in the intercellular space in the pellets. Chondrocytes in both pellets and explants exhibited extensive rough endoplasmic reticulum and Golgi complex (a, pellet, …

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Discussion

In direct comparison with native cartilage, this study characterized neocartilage generated by chondrocytes deriving from hyaline cartilage in pellet culture. After 1–2 weeks in culture, the neocartilage shared similarities with native cartilage with regard to chondrocyte phenotype, matrix distribution and the ultrastructure of collagen fibrils.

It has been suggested that an important consideration in engineering functional cartilage is to follow the principles of embryonic chondrogenesis (Solchaga et al. 2001). A critical step in chondrogenesis is the condensation of mesenchymal cells, which initiates cell–cell communication and leads to the birth of chondrocytes at the early stage of embryonic development (DeLise et al. 2000). Chondrocytes in the current study were condensed from a cell concentration of 1.0 × 10⁷ mL⁻¹ to a semisolid pellet. Chondrocyte density in the pellets increased two-fold over that in the explants. In the pellet, the close spatial relationship of neighboring chondrocytes and limited diffusion of newly formed matrix offer a better environment for cell–cell and cell–matrix communication (Farquharson & Whitehead, 1995) and facilitate regulation of chondrocyte differentiation (Hickok et al. 1998).

Retention of chondrocyte phenotype by the pellet culture was confirmed from the highly comparable staining patterns of immunohistochemistry for type II collagen, aggrecan and type IX collagen in the pellets with those in the cartilage explants. Type II collagen and aggrecan are tissue-specific markers for regenerated cartilage (Stewart et al. 2000; Anderer & Libera, 2002; Schulze-Tanzil et al. 2002) and are essential in distinguishing between hyaline cartilage and fibrocartilage, which has been responsible previously for the failure to generate a durable cartilage repair (Nehrer et al. 1999; Hunziker, 2001). At the same time, type I collagen synthesis was almost negligible in both pellet and explant culture. This also supports our notion that pellet culture is generating hyaline cartilage and not fibrocartilage.

The in vitro environment induces a much broader range of changes in chondrocyte dedifferentiation than previously thought (Schnabel et al. 2002). Type IX collagen, one of the fibril-associated collagens, has a role in connecting type II collagen and aggrecan (Olsen, 1997). In one study, the expression of type IX collagen was lost in dedifferentiated chondrocytes after monolayer culture, and did not recover when chondrocytes redifferentiated in alginate beads and re-expressed type II collagen and proteoglycan (Zaucke et al. 2001). This makes type IX collagen a sensitive marker of the tissue culture-associated redifferentiation of chondrocytes. In pellets, type IX collagen was steadily expressed through the 14 days of culture. Even at day 1 of pellet culture, when type II collagen and aggrecan were not detectable by immunohistochemistry and electron microscopy around the isolated chondrocytes, type IX collagen was immunohistochemically detected. Except for the conventional type II collagen and aggrecan, it is imperative to introduce additional markers for chondrocyte phenotype, such as type IX collagen, to monitor the process of cartilage engineering.

Active matrix production by chondrocytes in the pellets is evident by the remarkably increased size of the neocartilage and, at the same time, a gradually decreased cell density in the pellets during the culture period. The aggrecan concentration determines cartilage compression characteristics, while collagen fibril density, orientation and cross-linking determine its tensile property (Hasler et al. 1999). Scattered matrix macromolecules were observed in the pellet cultures as early as day 3. Quantitative immunohistochemistry for aggrecan indicates that aggrecan deposition by chondrocytes in the pellets quickly reached the level of cartilage explants during the period from day 3 to day 7, and it was maintained at virtually same level as in the explants up to day 14. This involved a continuous process of reconstruction of the fibril network in the pellets, as revealed by electron microscopy. By 2 weeks, the fibril meshwork in pellets was comparable with that of the explant cartilage. Several lines of evidence suggest that collagen fibrils developing in the pellets are similar to those found in native cartilage. Fibril diameter was not statistically different between the pellets and explant cartilage. Fibril density in pellets appeared lower than in explants, but this difference was also not statistically significant. However, it should be pointed out that even at day 14 the fibril orientation in the pellets did not have the same pattern as in native cartilage. The inferiority of the fibril network organization in the pellet-generated cartilage may ultimately impact on its biomechanical properties. One possible reason for the difference between the fibril network of the pellets and explants could be that there is a lack of mechanical stimulation during pellet culture; such physical stimulation exists during embryonic chondrogenesis of the explant cartilage. Combining mechanical stimulation with pellet culture could improve the fibril network because chondrocytes adapt to mechanical forces and remodel the organization of collagen fibres accordingly (Hasler et al. 1999), and also accelerate matrix production (Zhang et al. 2002).

In the model of cartilage regeneration we have described, the collagen fibril network formed between days 3 and 7, at which time the average intensity of aggrecan in the pellets reached the level seen in explants. This observation suggests that this period may be the optimal time for modulation of cartilage formation. By day 14, chondrocyte maturation was shown in the neocartilage, as indicated by the increased cellular volume. Although the marker for chondrocyte hypertrophy, type X collagen, was lacking, chondrocytes may proceed to hypertrophy quickly at this stage (Hirsch et al. 1996). When to terminate the culture to avoid chondrocyte hypertrophy is an important issue to consider, which has not been widely discussed in the field of tissue engineering.

We note that although the current study provides data on embryonic chick cartilage, certain clinical cartilage repair protocols are likely to be based upon the growth of mature human chondrocytes. Further investigation is required to specify appropriate growth characteristics in this context.

Conclusion

Hyaline cartilage was engineered by pellet culture of chondrocytes modelled after mesenchymal cell condensation in chondrogenesis. The pellet culture system retains the chondrocyte phenotype, and the resultant cartilage is therefore produced without the transformation of cell phenotypes or the need for scaffolding materials. Further interventions, such as mechanical stimulation, may be necessary to optimize the organization of collagen fibril network in pellet culture-generated cartilage.

Acknowledgments

This study was funded by the NIA Intramural Research Program, National Institutes of Health. We thank Dr Greta M. Lee for her help in pellet culture.

Want To Grow Taller? Let’s Get Started…

Hi,

My name is Michael. If you found this website you are probably interested at learning how to increase your height. I was once obsessed with this one goal and it consumed my thoughts for a good portion of my years and I have decided to create this website/blog in documenting my research in finding some type of solution or breakthrough in figuring out how to possibly increase human height both…

After the natural growth process ends when the epiphyseal plates close, and…
Before the natural growth process ends, when a person is still growing in height.

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You can check out the “Thoughts” post listings in the “Special Posts” but that is not necessary. Everyone has their own opinions, beliefs, and viewpoints and mine are just mine.

What else have we been researching and working on…

There are two sections below the main section entitled “Mind Hacks” and “Body Hacks” both of which is now integrated into the “Special Posts” section. These were posts I wanted to write up which I thought were really good tips and techniques to help the person improve themselves more than just their height. They are the most useful and unique ideas and methods I have ever read or heard about.

So what have types of research have we already looked into?

In the section “Techniques” you can see just what are the range of ideas that people have thought up over the years and tried to increase their height. The list of ideas and technique is long and you might be quite surprised at how many possible ways there are. Most of them are not very developed. The failure rate is very high and the feasibility of the ideas are very low. You can spend some time looking at what some of the ideas are but you don’t have to spent that much time since I can tell you guys right now that “there is no completely effective technique or methods found yet which can increase height in physically mature humans without invasive, expensive, and painful surgery which is also scientifically valid in theory and experiment.“

Check out the “All Posts” tab and scroll through the list of posts and articles I have wrote. Many of them are low in depth but only skim the surface of the subject. The posts I wrote from earlier on I would say is low on quality of scientific rigor but they get more and more advanced as the site progressed. If one of the articles catches your eye, take a look.

If you want to better understand the technical details on the research my team and I are looking into…

The fastest way to catch up really quickly on the subject is to go to “The Library” section and just skim through the hundreds of articles, patents, books, and scientific journals we have been collecting for the last few months

You will need to learn the fundamentals of at least 6 main areas of study to get a grasp on what areas of the body control the growing process. We want to move from the macro level down to the micro level. There is in order from top to bottom…

[Note: This section of 6 main technical subjects are still in development and A LOT of editing, adding of content, and changes will be done over time to improve the website.]

1. Orthopaedics,
2. Endocrinology,
3. Complete Height Increase Protein, Hormone, Compound, And Molecule Listing And Pathway Map,
4. Molecular Biology, Biochemistry, And Genetics,
5. Gene Database,
6. Biotechnology And Engineering

To find out what resources are available right now to reference and utilize …

It might be good to familiarize oneself with what tools, resources, and people one can get in touch with and use in this search. In the “Resources” section, there is a section which shows you a list of all of the posts that has ever been written on the website. In the list, you will find cool mind and body hacks which you can use and apply in your own life in case you would like to improve in other areas of life besides just height.

Listen to me and other guests talk about our latest research…

In the “Podcast” section I plan to do a weekly or biweekly podcast where I talk about any topics which might be related to the endeavor of height increase, the science of auxology, medical pathologies, and other related subjects. I will try to find medical specialists and height increase experts but I would think it might be a very hard task to get them to be willing to go on the air and share their voice and opinions.

Note: There is a “Forum” section to the website but that webpage is not activated at this time due to a personal judgement call made after discussing over the issue with other members of the community. Further considerations will be made in the future.

Be Informed of all the scams and frauds out there…

In the “Scams List” section is a large collection of the names and products of products which are found on the internet which sell fake products or services which don’t work. Maybe there is a chance they might be effective for young children who are still growing but for the majority of the adults who are looking for a solution, they are just useless. Take a look at the lists and be informed on what is out there waiting to take your money.

I wish you all success in your height increase endeavors.

Sincerely,

Michael

Epilogue…

I have been getting a few emails recently which expressed their frustration and confusion over the fact that they can’t seem to be able to figure out what to do. They come to this website wanting to learn more about how to possibility increase in height and grow taller but the general problem is that this website is either too large, too complex, or too advanced in the science for them to understand what the going on.

I think I am finally realizing that I can’t do this entire project by myself. I thought that I could but it is far larger of a problem than expected. I need some help from collaborators, people who can step up and take action to push this thing further then just what I or the few others have done so far.

There is no way I can dedicate so much time to this project, as well as run my primary business company, as well as attend school, and still be able to accomplish other responsibilities. I also realized that if something happened to me (god forbid), there is no way another person can take up the task and continue on exactly where I left off. They would have to go back and redo all the research.

This is my way (and strategy) of distributing the work load out by making it easier for anyone who is really passionate and dedicated to the idea of height increase and want to quickly catch up on the necessary information and research so that they can make a contribution in research and possible make breakthroughs and innovation.

One of my biggest frustrations was trying to go back to find what my predecessors have already done, tried out, and proven ineffective so that I can know what to not put my energy towards and save some time in searching. Some of them kept good records but most ideas and techniques are randomly scattered across the internet space. I am going to try to make this easier for the people who come after me.

Podcast

This page will be for the posting of the podcasts that will be done. That’s right, I’m starting a podcast. The episodes will be uploaded to the iTunes library and you can search for it. You will be able to download it to your apple products and play them when you are going about your day.

In the podcast, I will be focusing on the science of auxology, the different proposed techniques and methods of height increase, and relaying any recent news or scientific studies or article which might be relevant for the reader. I will of course be bringing in special guest which will probably include actual doctors, specialists, height increase researchers and enthusiasts, geneticists, and the average teenager or adult who wishes to grow taller and want to tell there story. Pretty much every related subject will be discussed.

I was recently informed of this idea to reach a larger audience through using the Podcast + Youtube method from listening to a Smart Passive Income Podcast by Pat Flynn and it got me inspired to take more action and get the message out to more people.

The process for me to apply and get accepted into iTunes as a real legitimate podcast may take me a few weeks so be patient about this endeavor. I don’t know how my voice sounds and I don’t listen to my voice a lot but no one I’ve ever met have said that I have a horrible voice yet so that is kind of encouraging.

Hope you are around for the first few episodes.

Click Here to Subscribe via iTunes and/or leave a review for the podcast!

Episode #1: For the first one, it is where I introduce myself and explain my story, where I come from, why I am doing this project. I then explain the few areas of research which seems the most promising. Length of time: 16 mins – Link Here –

Episode #2: Tyler from HeightQuest.com will be here to share their story, the type of research they have done, and what they think this field of research will be going towards in the coming yearss. Length of time: 31 mins – Link Here –

Episode #3: I take care of some website operation issues and then lead the conversation into a relatively short look and opinion of four topics which I have repeated talked about on the website. Length of time: 15 mins – Link Here –

Episode #4: This is part marketing and part manifesto to explain the reason why the venture of height increase is a high value and is a worthy cause to do research in. Most of it is my own perspective on the project. Length of time: 19 mins – Link Here –

Episode #5: Guest Hakker from GrowTallForum.com give his story, the research he originally was doing, and where he is at now. Along the way he gives his opinions on using growth plate transplants, the effectiveness of LSJL, Alkoclar, and more. Length of time: 36 minutes – Link Here –

[Update 12/30/2012 for Episode #5: Due to the interviewee’s request, this episode was taken down. The link no longer works. ]

Episode #6: For this episode I will be talking alone about details on the updates which are going on with the website, personal changes and how they will affect the evolution of the website, and projects and ideas which I want to combine with the website. Length of time: 16 minutes – Link Here –

Episode #7: Guest Tyler comes back to discuss more about LSJL, Gene Expression, Periosteum Manipulation, and we take a look at the best and most interesting articles and studies we’ve found in recent months and share our opinions and analysis on them. Length of time: 50 minutes – Link Here –

Episode #8: Guest Joel come on to discuss his lifelong desire to become taller and the psychology on why this desired has stayed with him over the years. We look at how this desire got started and stayed with him throughout the years. Length of time: 47 minutes – Link Here –

Episode #9: Guest Thomas Samaras From Reventropy Associates And HumanBodySize.com Discusses His Research On Human Stature. We go into the subjects of what his decades of research has shown about how human size and height is correlated with our longevity. Length of time: 50 minutes – Link Here –

Episode #10: I Talk About The Research And Science Behind Adult Height Increase Blog. I go into the subject of the old Monster BS Formula and the new PTH Formula used to deossify bones and increase stem cell proliferation. Also mention the fact that growth plates have been grown. Length of time: 30 minutes – Link Here –

Episode # 11: I Review And Outline All The Research And Studies Matheus Has Shown To Me. Some of the stuff he has found has definitely helped in clearing up a few blind spots me and Tyler have had in our own research. Length of time: 26 minutes – Link Here –

Episode #12: Our Guest Andrew comes on to talk about his experiences in getting limb lengthening surgery. He went to Dr. Betz and had his femurs extended using the internal method by almost 4 inches increasing his height. Length of time: 41 minutes – Link Here –

Tools Used

Music Used: One amazing tips that Pat Flynn gave me from watching his Podcast Series (Video #2) on Youtube HERE is that when I start out the podcast, I should use what are known as “Royalty Free Music” because that is the type of music that I can use without getting into trouble with copyright laws and legal problems. The music I have chosen to begin and end all of my podcast episodes is by Johann Sebastian Bach Suite No. 6 & Mozart – Sonate No. 12, 13. I don’t think two guys who have been dead for over 200 years can sue me. You can find the original link to the entire song from MusOpen.Org. I like it.

Due to the low volume of the music from the last two episodes, I have decided to switch the intro and ending music up for something that is slightly louder. The music that I decided to use for this episode (#3), and probably subsequent episodes to come, is entitled” Air on the G String” performed by the US Air Force Band using the String Orchestra instruments and Symphonic Pieces form. The original composer is Johann Sebastian Bach from the Baroque period . You can get the music Mp3 track at MuseOpen.Org which provides a source where you can download non-licensed, non copyrighted music which you can use for one’s purpose. The specific link is HERE.

Software & Media Host Used: From listening to other podcasters, I chose to use the service of Libsyn.com. I decided to go for the package where I pay $15/mth for the ability to upload up to 250 MB of media file size every month.

Recording Software & Hardware Used: Since I own a 20112 13″ Macbook Air I just use the available application GarageBand already installed on my laptop to record and upload all of my mp3 files to the itunes directory. You can find my podcast by typing in “Natural Height Growth” into the itunes directory, library, or store (not sure which one yet). As for a microphone voice recorder, I am using either an iphone recorder (the small thin white ones) or using the “Voice Recorder” app from the Apple “App Store”. I think it is free. From listening to Cliff Ravenscraft’s Podcast Tutorial Series HERE (Part 3 of 8), I know that my voice quality is not that good right now, but if my podcast has a good number of listeners, I’ll switch the voice recording hardware equipment for something better.

To record the podcast episodes where a guest come along, I used Skype to take the actual voice calls but use a software to record Skype calls called “Call Recorder, For Skype” from Ecamm.com. This cost $20 to get.

Music Player & WordPress Plugin Used: To put the podcast easy to reach and use, we chose to use a WordPress plugin called Blubrry PowerPress. This plugin ia also great to be used as a feed

My Focus On The Main Business And Company

A small note to the people who come here regularly.

I’ve been putting a lot of time and energy into the this project for the last 4 months which has been a wonderful time which allowed me to fulfill a passion of mine. I now have to shift my focus on the other projects and responsibilities in my life. Currently there are things I have to do for my company which is my main business, my other online business, my school obligations, and my obligations for the people around me. I plan to put maybe another 2-4 full months of intense work and research on this website and then lessen the rate of research.

I was hoping from the beginning that I could find and assimilate all of the possible information from the internet on height increase to this website and I think that the task is mostly achieved. Any further research would probably be where I try to assimilate all the facts and research together to come up with new ideas and breakthroughs.

Buying GH From Sino Human Growth Hormone Pharmaceutical

[Warning: Don’t start buying and using Growth Hormones hoping to increase your height unless you are absolutely certain that it is safe (or maybe just don’t care). GH is not recommended or even allowed legally into certain countries for people who are still growing and GH will not work for people who can take them legally which is after growing stops. It is sort of like a catch-22 people get stuck in. The only way legally to get around the loophole is if a person can get a specialist endocrinologies tho administer it to them clinically.]

I have recently become aware that there seems to be quite a few suppliers around the world which can get you synthetic Growth Hormones. Obviously the problem is that sometimes these suppliers don’t get you real GH but fake stuff.

There is a company from South China Sino Human Growth Hormone Pharmaceutical Co., Ltd which seems to sell almost everything. It is really hard for me to tell whether they sell the real GH stuff or the fake stuff. There are a few places where buyers and users review them but they are rather mixed.

From the website Eroids.com, most people who comment say that the company has “legit stuff” and has “good quality”. There seems to be even a linkedin account for Sino HGH and and the managers there and more. from the linked in account…

“Sino Co., Ltd is a company which specialize in human grow hormone,anabolic steroids,and peptides. We have own lab to produce finished prdocuts.
Sino Human Growth Hormone Pharmaceutical Co.,Ltd.
We aim to be the best and the largest HGH, Injectable Steroids, oral steroids, anabolic steroids powder and Peptides supplier in China. Our company is located in Shenzhen since 2003, we gained a great reputation among our customers because of our professionalism in United States, United Kingdom, Europe,South Africa,South Asia,Latin America,etc.
Best quality, fastest delivery,safest packing,most timely
aftersales service are the keys that we got credit from our clients all over the world. It is our ultimate goal to extend market presence to every part of the world. We warmly welcome sincere cooperation with customers from at home and abroad to explore a bright future together.”

This is the price listing they have…

Price list of all products from Sino HGH Pharm Co.Ltd

Human Growth Hormone Series:
Generic HGH——– $115/kit
Blue Top 4IU ——– $75/kit
Kigtropin ——– $120/kit
Jintropin ——– $180/kit
Hygetropin ——– $420/kit
Ansomone ——– $200/kit
Taitropin ——– $120/kit
Igtropin ——– $70/kit
IGF1 LR3 1mg ——– $70/kit
IGF1 LR3 10mg ——– $320/kit

Injectable anabolic steroids Series:
Testosterone Propionate ——- $40/kit
Testosterone Enanthate ——– $45/kit
Testosterone Cypionate ——– $45/kit
Sustanon 250 ——– $60/kit
Nandrolone Decanoate ——– $65/kit
Boldenone Acetate ——– $65/kit
Boldenone Udecylenate ——– $65/kit
Winstrol/Stanozolol ——– $45/kit
Trenbolone Acetate 75 ——– $70/kit
Trenbolone Enanthate 100 ——– $75/kit
Primobolan ——– $78/kit
Masteron ——– $68/kit
Testosterone Undecanoate ——– $33/kit
Erythropoietin (EPO) ——– $60/kit
Menotrophinfor Injection (HMG) ——– $60/kit
H C G ——– $65/kit

Specialties

Sino peptides series:

PT-141 ——– 110$/kit
MT-2 ——- 110$/kit
GHRP-2 ——– 80$/kit
GHRP-6 ——- 80$/kit
CJC-1295 ——- 105$/kit
Sermorelin ——– 110$/kit
Ipamorelin ——– 110$/kit
Hexarelin ——– 110$/kit
MGF ——– 90$/kit
Peg-MGF ——– 250$/kit

One thing I am not sure is whether it is possible that good steroids and peptides can go bad if the inventory is shelved for too long like food stuff. If that is case maybe the stuff the Chinese company was originally selling was good but went bad.

The price range for the steroids and peptides, which include many types of steroids which I have researched for possible height increase possibilities are quite reasonable.

The cost of shipping is from $45-$100 assuming the stuff is being bought by people in the US and UK. The amount of time you have to wait is usually 7-10 business days. They only accept Western Union, money grams, and bank transfers, but no Paypal or other escrow methods. (taken from FAQ section) The success in the delivery is not very high for certain countries.

Of course you always have the contact information below for any types of inquiry or concerns.

Contact Us

Name: Miss Wendy Tang
Skype: sinohgh
MSN: sino-hgh-01@live.com
E-mail: sino-hgh-01@live.com

Contact Information

Company Name:	Sino Human Growth Hormone Pharmaceutical Co., Ltd
Contact Person:	Jack Liu (Your message will be sent to the supplier directly)
Address:	#735-740,Yingfeng Valley,Chuangchun Road, nanjing, Jiangsu, China
Telephone:	86-135-1072-1924
Fax:	86-135-1072-7895

While I don’t plan to be taking the GH injections anytime soon since it won’t really help increase height, it might be a good place for us to buy GH in case we are interested in doing a few studies and experiments. If a young person is crazy and desperate enough, I probably would point to this company to use so that they at least don’t get the fake stuff which can hurt them.