Biochemical and physical signal gradients in hydrogels to control stem cell behavior.

“Cells continuously receive biochemical and biophysical stimuli from their microenvironment. These environmental stimuli drive cellular behavior and function during development and tissue regeneration.”<-We can alter the microenvironment via mechanical means to alter cellular behavior and ideally create neo-growth plates.

“Cell interactions with the extracellular matrix (ECM) and physical signals such as matrix rigidity and mechanical stimuli can also have strong effects on cellular phenotype and tissue formation.”<-The bone ECM likely has a strong negative regulatory effect on neo-growth plate formation.  But we can alter the bone ECM via mechanical means as well.

“[we prepared] HP-ALG hydrogels with incorporated gradients of heparin binding growth factors BMP-2, a potent osteogenic growth factor, and TGF-β1, a chondrogenic growth factor, in opposite directions. When the concentration of growth factors in segments of gradient HP-ALG hydrogels was quantified, linear gradient distributions of BMP-2 and TGF-β1 in opposite directions were observed”

“As the BMP-2 concentration increased, ALP expression significantly increased along the BMP-2 gradient. In contrast, GAG production of encapsulated hMSCs significantly increased as the TGF-β1 concentration increased”

Regulation of Stem Cell Fate in a Three-Dimensional Micropatterned Dual-Crosslinked Hydrogel System.

“By manipulating micropattern size while keeping the overall ratio of single- to dual-crosslinked hydrogel volume constant, the physical properties of the micropatterned alginate hydrogels are spatially tunable. When human adipose-derived stem cells (hASCs) are photoencapsulated within micropatterned hydrogels, their proliferation rate is a function of micropattern size. Additionally, micropattern size dictates the extent of osteogenic and chondrogenic differentiation of photoencapsulated hASC. The size of 3D micropatterned physical properties in this new hydrogel system introduces a new design parameter for regulating various cellular behaviors, and this dual-crosslinked hydrogel system provides a new platform for studying proliferation and differentiation of stem cells in a spatially controlled manner for tissue engineering and regenerative medicine applications.”

“cell behaviors such as differentiation and proliferation are known to be affected by cell cluster size”

” micropattern size dictated the extent of osteogenic and chondrogenic differentiation of photoencapsulated hASC.”

“the aggrecan expression of hASCs gradually increased as the micropattern size increased”<-But some markers increased at 200 micrometers so the optimal micropattern size should be around 100-200 micrometers.

HEre’s a visualization of micropatterns:

Decellularized tissue and cell-derived extracellular matrices as scaffolds for orthopaedic tissue engineering.

“The reconstruction of musculoskeletal defects is a constant challenge for orthopaedic surgeons. Musculoskeletal injuries such as fractures, chondral lesions, infections and tumor debulking can often lead to large tissue voids requiring reconstruction with tissue grafts. Autografts are currently the gold standard in orthopaedic tissue reconstruction; however, there is a limit to the amount of tissue that can be harvested before compromising the donor site. Tissue engineering strategies using allogeneic or xenogeneic decellularized bone, cartilage, skeletal muscle, tendon and ligament have emerged as promising potential alternative treatment. The extracellular matrix provides a natural scaffold for cell attachment, proliferation and differentiation. Decellularization of in vitro cell-derived matrices can also enable the generation of autologous constructs from tissue specific cells or progenitor cells. Although decellularized bone tissue is widely used clinically in orthopaedic applications, the exciting potential of decellularized cartilage, skeletal muscle, tendon and ligament cell-derived matrices has only recently begun to be explored for ultimate translation to the orthopaedic clinic.“

“ECM is a product of cells that functions to maintain tissue and organ structure, organization and function. It is a complex network of proteins and polysaccharides forming an intricate meshwork within tissue that interacts with the resident cells to regulate cell behavior, such as migration, proliferation and differentiation. The ECM exists in a state of dynamic equilibrium with its resident cells and is constantly being built, reshaped and degraded in response to changing environmental conditions and to cellular, tissue and organ demands”<-So we should try to alter the bone ECM to be more favorable to cartilaginous tissues.

“Fracture healing requires an intricate and well-organized series of cellular and molecular events. It involves interactions between cortical bone, the periosteum, undifferentiated fascial tissue surrounding the fracture and the bone marrow. Fracture healing is divided into three stages: inflammation, repair and remodeling. After an injury, there is initial bleeding from the damaged bone ends and surrounding tissue resulting in the formation of a hematoma, which provides a source of hematopoietic cells capable of secreting growth factors. The invasion of inflammatory cells, fibroblasts, mesenchymal cells, and osteoprogenitor cells at the fracture site forms granulation tissue around the fracture ends{To induce neo-growth plates we have to allow this invasion}. Fractures that are anatomically aligned with absolute stability, such as those surgically repaired with compression plates, undergo primary bone healing or Haversian remodeling, in which there is direct osteonal healing within the cortex by intramembranous ossification”

” in closed reduced fractures, secondary bone healing occurs with the formation of a bridging soft callus consisting of cartilage tissue connecting the fracture ends. Over time, bone formation occurs under the periosteum and calcification of cartilage results in the formation of hard callus or woven bone by endochondral ossification”

“injuries that penetrate the subchondral bone often result in the formation of fibrocartilage which is biomechanically insufficient compared to hyaline cartilage, resulting in further damage over time”

“the peak force transmitted through the Achilles tendon while running is 9 kN, which is about 12.5 times the body weight”

A sesamoid is a bone formed within a tendon or muscle while we’re looking for is more interosseous chondroification(the formation of new cartilage tissue within the bone) or probably maybe even first interosseous epithelialification as you might need to transition mesenchymal to epithelial tissues first.

Coincident development of sesamoid bones and clues to their evolution.

“Sesamoid bones form within tendons in regions that wrap around bony prominences. They are common in humans but variable in number. Sesamoid development is mediated epigenetically by local mechanical forces associated with skeletal geometry, posture, and muscular activity. In this article we review the literature on sesamoids and explore the question of genetic control of sesamoid development. Examination of radiographs of 112 people demonstrated that the relatively infrequent appearances of the fabella (in the lateral gastrocnemius tendon of the knee) and os peroneum (in the peroneus longus tendon of the foot) are related within individual. This finding suggests that the tendency to form sesamoids may be linked to intrinsic genetic factors. Evolutionary character analyses suggest that the formation of these sesamoids in humans may be a consequence of phylogeny. These observations indicate that variations of intrinsic factors may interact with extrinsic mechanobiological factors to influence sesamoid development and evolution.”

“As many as 42 sesamoid bones can be found in some individuals. Mechanically, sesamoid bones serve to protect the tendon from damage and, in some cases, increase the efficiency or mechanical advantage of their associated muscle.”

“Most sesamoid bones in humans are 5 to 10 mm in diameter or smaller and are present in 1 to 100% of individuals.”

“Tendon chondrometaplasia[tendon turns into cartilage] and sesamoid bones tend to develop within tendons in areas that experience both tensile strain and hydrostatic compressive mechanical stresses.”<-So tendonous tissue may help form neo growth plates.  Tendon enthesis attach into the bone.  Tendon is capable of undergoing  chondrometaplasia.  So potentially you could form neo growth plates at these tendon entheses.  Originally I thought that ligament enthesis had more potential for neo-growth plate formation but tendon enthesis may have potential too.

” fibrous tendon tissue can form regions of fibrocartilage in areas that wrap around bony prominences (fibrocartilage is a tissue whose phenotype is intermediate between fibrous and cartilaginous tissue, consisting of chondrocytes embedded in aligned bundles of type I collagen)”

“compression loading and treatment with transforming growth factor beta (TGF-β) each resulted in upregulation of aggrecan and biglycan synthesis in fetal bovine tendon, suggesting that one aspect of the response of cells to compressive load is increased TGF-β synthesis which, in turn, stimulates synthesis of extracellular matrix proteoglycans and leads toward fibrocartilage formation. The process of fibrocartilaginous metaplasia in tendons, which is a direct response to an altered mechanical loading environment, appears to represent an intermediate step in the formation of a sesamoid cartilage.”

Sesamoid bones:

Their position won’t help you grow taller but they could if they were in the articular cartilage.  Maybe if tendonous tissue was inserted in the articular cartilage.

“Hox A11, had profound effects on the developing mouse skeleton, including abnormal sesamoid bone development in both the forelimbs and hindlimbs.”

How might we turn tendons(and especially tendenous enthesis) into cartilage tissues.  What if we form a sesamoid bone within the enthesis?

Expression of Bone Morphogenetic Protein-2 in the Chondrogenic and Ossifying Sites of Calcific Tendinopathy and Traumatic Tendon Injury Rat Models

“Ectopic chondrogenesis and ossification were observed in a degenerative collagenase-induced calcific tendinopathy model and to a lesser extent, in a patellar tendon traumatic injury model. We hypothesized that expression of bone morphogenetic protein-2 (BMP-2) contributed to ectopic chondrogenesis and ossification. This study aimed to study the spatial and temporal expression of BMP-2 in our animal models.
Seventy-two rats were used, with 36 rats each subjected to central one-third patellar tendon window injury (C1/3 group) and collagenase-induced tendon injury (CI group), respectively. The contralateral limb served as controls. At week 2, 4 and 12, 12 rats in each group were sacrificed for immunohistochemistry and RT-PCR of BMP-2.
For CI group, weak signal was observed at the tendon matrix at week 2. At week 4, matrix around chondrocyte-like cells was also stained in some samples. In one sample, calcification was observed and the BMP-2 signal was observed both in the calcific matrix and the embedded chondrocyte-like cells. At week 12, the staining was observed mainly in the calcific matrix. Similar result was observed in C1/3 group though the immunopositive staining of BMP-2 was generally weaker. There was significant increase in BMP-2 mRNA compared to that in the contralateral side at week 2 and the level became insignificantly different at week 12 in CI group. No significant increase in BMP-2 mRNA was observed in C1/3 group at all time points.
Ectopic expression of BMP-2 might induce tissue transformation into ectopic bone/cartilage and promoted structural degeneration in calcific tendinopathy.”

“the presence of chondrocyte phenotype and ectopic ossification in a collagenase-induced patellar tendon injury model”

Here’s potential chondrogenic lesions:

What can we discern about the plans from LSJL from the grants?  Yokota is one of the primary scientists behind the study Lengthening of Mouse Hindlimbs with Joint Loading.  There does not seem to be very much on the LSJL length effects since the expiring of Ping Zhang’s 2010 grant.  We either have to study the lengthening effects on our own or help Ping Zhang get more funding.

Yokota doesn’t mainly study the lengthening effects which are primarily studied by Ping Zhang as shown by this grant.

Here’s Hiroki Yokota’s 2015 grant:

“The long-term objective of the proposed studies is to elucidate the mechanism of mechanotransduction in bone. Our present bioengineering-oriented project developed a high-resolution piezoelectric mechanical loader and evaluated the role of mechanical stimulation in bone using cultured osteoblasts. The results reveal that (a) deformation of 3D collagen matrix can induce strain-induced fluid flow;(b) strain-induced fluid flow, and not strain itself, predominantly activates the stress-responsive genes in osteoblasts;and (c) architecture of 3D collagen matrix establishes a pattern of strain-induced fluid flow and molecular transport{We are not interested so much on the effects on osteoblasts but more on the effects of fluid flow on bone degradation and fluid flow on mesenchymal stem cells to create neo growth plates}. Many lines of evidence in animal studies support enhancement of bone remodeling with strain of 1000 – 2000 microstrains. An unclear linkage between our in vitro studies and these animal studies is the role of strain and fluid flow in bone remodeling. In vitro osteoblast cultures including our current studies use 2D substrates or 3D matrices that hardly mimic the strain-induced fluid flow in vivo. This difference between in vitro and in vivo data makes it difficult to evaluate the role of strain and fluid flow in bone remodeling and anti-inflammation. First, microscopic strain in bone might be higher than the macroscopic strain measured with strain gauges. A local microscopic strain higher than 1000 – 2000 microstrains may therefore drive fluid flow in bone. Second, the lacunocanalicular network in bone could amplify strain-induced fluid flow in a loading-frequency dependent fashion{This we should try to modify the frequency of LSJL to amplify strain}. Lastly, interstitial fluid flow in bone might be induced by in situ strain as well as strain in a distant location, such that deformation of relatively soft epiphyses induces fluid flow in cortical bone in diaphyses{We are more interested in deformation of the epiphysis as that’s where growth plates typically occur but deformation of the epiphysis in one end may induce fluid flow in the epiphysis in the other}. This renewal proposal will use mouse ulnae ex vivo as well as mouse in vivo loading to examine the above possible explanations for the data divergence.
Specific aims i nclude: (1) fabricating a piezoelectric mechanical loader for ex vivo and in vivo use;(2) quantifying ex vivo macroscopic and microscopic strains using electronic speckle pattern interferometry as well as molecular transport using fluorescence recovery after photobleaching;(3) conducting bone histomorphometry to evaluate ex vivo data;and (4) examining load-driven adverse effects with gene expression and enzyme activities (e.g., matrix metalloproteinases). Mechanical loads will be given in the ulna-loading (axial loading) and elbow-loading (lateral loading) modes{he’s planning on doing another LSJL loading study!}. These two modes have been shown to enhance bone remodeling in the diaphysis with different patterns of strain distribution. Successful completion of the proposed renewal proposal will provide basic knowledge about induction of fluid flow in bone and establish a research platform for devising therapeutic strategies for strengthening bone and preventing bone loss.”

Here’s the Hiroki Yokota 2014 grant:

Mechanical Loading and Bone

“The long-term objective of this study is to elucidate the mechanisms underlying loading-induced bone remodeling and develop unique loading-based therapies for preventing bone loss. The specific goal of this study, based on our most recent observations, is to determine how mechanical loading to the knee (knee loading – application of mild lateral loads to the knee) may exert global suppression of osteoclast development not only in the loaded (on-site) bone but also in the non-loaded (remote) bone{This is unfortunately not very promising for height growth as osteoclast driven remodeling is pretty significant for growth plate formation}. As a potential regulatory mechanism, we will focus on secretory factors (e.g., Wnt3a, NGF?, TNF?, etc.) and low-density lipoprotein receptor-related protein 5 (Lrp5) mediated signaling. In the parent project, we have shown that knee loading enhances bone formation in the tibia and the femur through the oscillatory modulation of intramedullary pressure. However, its effects on bone resorption have not been well understood. Preliminary studies using a mouse ovariectomized model, which mimics post-menopausal osteoporosis, indicate that knee loading can suppress development of multi-nucleated osteoclasts from bone marrow cells, and the loading effects are observed not only in the loaded femur but also in the non-loaded contralateral femur. In this competitive renewal project, we will test the hypothesis that joint loading (knee/elbow loading) can suppress an OVX-induced osteoclastogenesis in a systemic manner through Lrp5-mediated Wnt signaling with Wnt3a as a secretory factor, as well as interactions with other secretory factors. To examine this hypothesis, we propose two specific aims using a mouse loading model (knee loading, elbow loading, ulna bending, and tibia loading), and assays for bone remodeling and primary bone marrow cells.
Aim 1 : Determine the local and global effects of joint loading on osteoclastogenesis Aim 2: Evaluate the role of load-modulated secretory factors in osteoclastogenesis In response to mechanical loading, we will conduct X-ray imaging and colony forming unit assays{The x-rays will be highly useful in determining whether LSJL can induce neo-growth plate formation although the effects would have to be large to show up on the xray}. We will also examine expression of critical secretory factors such as Wnt3a, NGF?, TNF?, OPG, RANKL, etc. in the serum. Primary bone marrow cells will be cultured, and the mechanisms underlying loading-driven regulation of osteoclastogenesis will be investigated. We will examine expression of regulatory factors, including NFATc1 (master transcription factor for osteoclastogenesis) and osteoclast markers such as OSCAR, cathepsin K, etc. We will employ Lrp5 KO mice (global, and conditionally selective to osteocytes), as well as neutralizing antibodies and RNA interference (loss of a function), and plasmids (gain of a function). We expect that this project will contribute to our basic understanding of load-driven regulation of bone resorption and development of loading regimens useful for global prevention of bone loss. ”

Let’s look at Hiroki Yokota’s 2013 Grant:

“The long-term objective of the proposed studies is to elucidate the mechanism of mechanotransduction in bone. Our present bioengineering-oriented project developed a high-resolution piezoelectric mechanical loader and evaluated the role of mechanical stimulation in bone using cultured osteoblasts. The results reveal that (a) deformation of 3D collagen matrix can induce strain-induced fluid flow{If it is fluid flow that can induce neo-growth plate formation via stem cell simulation then we need to make sure that LSJL deforms the 3D collagen matrix};(b) strain-induced fluid flow, and not strain itself, predominantly activates the stress-responsive genes in osteoblasts;and (c) architecture of 3D collagen matrix establishes a pattern of strain-induced fluid flow and molecular transport. Many lines of evidence in animal studies support enhancement of bone remodeling with strain of 1000 – 2000 microstrains{2000 microstrain is about a 0.2% change in bone length.  LSJL laterally compresses the bone so the compression has to be by at least .1 or .2% to work}. An unclear linkage between our in vitro studies and these animal studies is the role of strain and fluid flow in bone remodeling. In vitro osteoblast cultures including our current studies use 2D substrates or 3D matrices that hardly mimic the strain-induced fluid flow in vivo. This difference between in vitro and in vivo data makes it difficult to evaluate the role of strain and fluid flow in bone remodeling and anti-inflammation. First, microscopic strain in bone might be higher than the macroscopic strain measured with strain gauges. A local microscopic strain higher than 1000 – 2000 microstrains may therefore drive fluid flow in bone. Second, the lacunocanalicular network in bone could amplify strain-induced fluid flow in a loading-frequency dependent fashion. Lastly, interstitial fluid flow in bone might be induced by in situ strain as well as strain in a distant location, such that deformation of relatively soft epiphyses induces fluid flow in cortical bone in diaphyses{of course our goal is to create new growth plates in the epiphysis but the fluid flow from compressing the ends of the epiphysis may flow deeper helping to induce mesenchymal condensation to induce neo growth plates closer to where the epiphysis meets the diaphysis}. This renewal proposal will use mouse ulnae ex vivo as well as mouse in vivo loading to examine the above possible explanations for the data divergence.
Specific aims include: (1) fabricating a piezoelectric mechanical loader for ex vivo and in vivo use;(2) quantifying ex vivo macroscopic and microscopic strains using electronic speckle pattern interferometry as well as molecular transport using fluorescence recovery after photobleaching;(3) conducting bone histomorphometry to evaluate ex vivo data;and (4) examining load-driven adverse effects with gene expression and enzyme activities (e.g., matrix metalloproteinases). Mechanical loads will be given in the ulna-loading (axial loading) and elbow-loading (lateral loading) modes. These two modes have been shown to enhance bone remodeling in the diaphysis with different patterns of strain distribution. Successful completion of the proposed renewal proposal will provide basic knowledge about induction of fluid flow in bone and establish a research platform for devising therapeutic strategies for strengthening bone and preventing bone loss. ”

The grants from 2013-2006 are virtually the same.  It’s only 2014 which is different however it’s unfortunate that it’s not focusing on the lengthening effects.

Unfortunately Ping Zhang’s grant Load-Driven Bone Lengthening only ran from 2008-2010.

There were a few people in recent days who wanted to start a KickStarter project to fund this type of project. The project is the idea of using regenerative medicine, stem cell therapy, tissue engineering, and 3D Bioprinting to get an alternative to limb lengthening surgery into market.

What they were discussing was a recent post I wrote entitled – “How Close Are We Towards Growth Plate Regeneration To Grow Taller?”

In response, I have to show these people just how crazy it is to get something new in medicine to the public. Refer to Forbes.com post written back in 2014 entitled – “The Truly Staggering Cost Of Inventing New Drugs”

Eli Lilly, states that it would cost on average about $1.3 Billion USD to get just one drug approved and into the market. $1 Billion Dollars!

If you then take into consideration just how easy it is to have drugs fail trials and testing, then the real cost could be around $4 Billion. The high range is $11 Billion for just one drug to be approved.

I did a quick search on Google to find out what is the project in Kickstarter’s history which got the most funding. It seems that based on this article in Entrepreneur.com it would be the Pebble Time based Watch, with a grand total of $20 Mil. $20 Mil is indeed a lot of money to fund such a project. Of course I have been to trade shows for the latest electronic gear in Hong Kong and China before so I have seen at least 4-5 companies who have also come out with similar type products, aka a smart watch which looks suspiciously similar in design to the Apple Watch.

What we would need to get this project started would be something at least 50X that amount. How many people would even believe us that this idea we have is even possible?

I have emailed someone named Harald from the beginning of this website back in 2012 and he has still not been able to raise even $100 K  to get people to fund what he says is research. He claims he is part of a biomedical team of researchers. It has been 10 years and so far no one has been willing to step up. The honest truth is that no one will step up. The first $50 Mil will be just wasted money, since it will just be used as initial startup cost, which will be sunk cost which would take maybe 20 years to get back, if the project is successful.

This idea I have shown to be quite valid would require a truly herculean amount of will, effort, and money to get it done. There is probably less than 10 people in the entire world who would have the financial ability and incentive to fund us. Of course, for any type of potential investor to invest in us, (since no one wants to loose money on a bad investment), we would have to first show very clearly that the science and the technical details are completely valid and it will work.

Trying to get people to open their wallets is one of the hardest things one learns to do as an adult. Selling is truly the most important skill to learn.

Instead of going on Kickstarter, me and Tyler would have to go on Shark Tank and ask Mark Cuban, who actually has a billion, and the other less rich sharks to give us at least $100 Mil to get started on this project. Of course, then once we reach the first goal, we would have to go back and ask for another $300 Mil. At $300 Mil, not even someone like Kevin O’Leary would have the money to fund our endeavor.

I can see Mark Cuban, who loves basketball and owns a basketball team being very interested in funding a company or project like this, as well as the other sharks. Unlike Cuban, who is 6′ 3″, the others are of short stature. Daymond John, Kevin O’Leary, Robert Herjavek, Barbara Corcoran, they are all on the shorter side. I can see a trip on Shark Tank being most likely to work out. It would at least give our project the big exposure we need, and the short statured “sharks” would definitely be interested in getting something in life which even they can’t buy with their millions. Money can buy limb lengthening surgery for these guys but they probably will never be willing to put up with the pain, loss of time, and helpless feeling.

The other options is to find a rich Arab/Saudi Sheik or Prince who might have a height complex and is willing to give us maybe $50-$100 Mil to get the project started. Before when I used to work in the alternative energy sector in a former life, the CEO at the time said that the company I was at was in talks with a Saudi Family in getting investments (as well as the billionaire T Boone Pickens). My CEO told in passing that apparently the real net worth of the Saudi Family was a total of $1 Trillion!! (True Story).

The third option is to look for a crazy billionaire who was a scientist or biologist in a former life. Off of the top of my head, I am thinking Patrick Soon-Shiong. Patrick is supposed to be the richest person in all of Los Angeles. He is the type of person who is willing to take moon-shots, similar in style to Google. At the current moment Soon-Shiong is willing to put his own billions in looking for a cure for different types of cancers. If he is willing to put billions down to search for a cure for cancer, he just might be willing to also put a billion down to get this stem cell therapy to work out. I honestly believe that if this form of limb lengthening surgery alternative reaches the public market, it will become a multi-billion dollar industry within 10 years.

The last option is just a crazy billionaire who is willing to believe in our idea. Richard Branson. From Necker Island. Is someone like Branson willing to talk to a person like me? Personally, I have met Branson before when I was in Las Vegas in an audience where he was giving a speech and Q&A. He was very inspirational but he is also a very smart, shrewd, calculated business man who is always willing to get a good deal on a business.

If we are going to present our idea to any of these billionaires, I suggest that first we actually really dig deep into the science, learn everything we can, write out a 1000+ page book out to explain all of the technical details, and find someone who knows these types of investors in maybe 5 years.

This is assuming that the researchers at EpiBone don’t come along and get further into the research and development than us.

Recent searching through the Google Patent Database revealed a few patents which have been filed which pertains to our goals.

The most important patent is a couple of researchers from Columbia University, Jie Jiang and Helen Lu entitled “Methods for inhibiting cartilage mineralization – WO 2008156725 A2″.

What makes this patent so unique and special is that the chemical that is proposed for injection to the locallized area is a chemical which I have written before multiple times and theorized was the key to possible bone interstitial growth even after bone maturity. – “Parathyroid Hormone And Parathyroid Hormone-Related Protein May Lead To Non-Invasive Epiphyseal Growth Plate Regeneration (Big Breakthrough)”.

Of course that post was written in late 2012, which was only the very start of my research. It seems that this patent which was filed back in 2008 seems to validate this idea that using Parathyroid Hormone-Related Protein (PTHrP) would have inhibitory effects on the mineralization process on chondrocytes in hyaline cartilage.

Here is what is important to realize. This patent technically talks about using PTHrP on articular cartilage. The fact that two researchers from a leading research university is willing to put 5,000K USD to file a patent for this idea shows that the science is real. They thought the idea was viable enough to get a patent on it. So I would say it is reasonable to assume that using PTHrP on the articular cartilage will prevent the chondrocytes from mineralization.

Scientifically speaking, when you do the research on looking at how IHH (Indian Hedgehog) controls the rate of stimulation of PTHrP in the profeliferation and hypertrophic layers of the growth plate, it might cause the more discriminatory researcher to suggest that maybe IHH, not PTHrP should be the chemical we should focus on. Technically, it is true that IHH is the chemical that will cause PThrP to be stimulated. There is a negative feedback look in the layer of the growth plate. If the PTHrP chemical is stimulated, it tells the levels of IHH to drop, like a sort of check system to make sure the chemical process doesn’t turn into a run-away chemical reaction chain. However, from what I remember, it is PTHrP that is what actually causes the type of chondrocyte actions that we wants, specifically increased proliferation and then increased hypertrophy. In addition, I seem to remember vaguely at least 1 study which says that increased PTHrP seems to prevent the hypertrophic chondrocytes from undergoing apoptosis too quickly.

This is why I believe that this patent (or method/technique) can be translated to the application of inhibiting chondrocyte mineralization also in the epiphyseal cartilage layers.

If you read the patent and dig into the details, the inventors mention that there is at least half a dozen ways to get the PTHrP to be administered to the deep zone layer of articular cartilage. if there is a half different way to get the chemical to reach the end of the bones/ epiphysis, then there is probably the same number of ways to reach a growth plate that is still not fully closed.

To further validate this idea, there was also another patent filed by a completely different research team based in China on the same idea. You inject PTHrP to stop early onset osteoarthritis.  Refer to the patent # “Treatment of early-stage osteoarthritis
US 8586533 B2″. For this particular 2nd patent, they prefer the intra-articular injection method. Also, for the exact amount of PTHrP used, they suggest within the range of from about 0.1 nM to about 200 nM in the synovial fluid of the synovial joint. 

What is even more amazing is that fact that we don’t even need to use this organic protein alone to inhibit chondrocyte mineralization.

Refer to the studies below.

• Collagen/annexin V interactions regulate chondrocyte mineralization.
• Annexin V and terminal differentiation of growth plate chondrocytes.
• http://www.sciencedirect.com/science/article/pii/S0945053X01001160
• TGF-beta1 is the factor secreted by proliferative chondrocytes to inhibit neo-angiogenesis.

Here are the other chemicals to stimulate. – TFG-Beta1, Glutamate, and Vitamin C Sulfate.

Here are the chemicals to decrease – Annexin V

So theoretically, would injection of a TGF-Beta1 and PTHrP combination into the layer of growth plates work in stopping the growth plates from completely closing up?

Of course, no chemical is good enough to completely inhibit the process of epiphyseal fusion. I forget which study I read (involving Sox9) which explained in extremely fine detail what was the exact chronological steps for the chondrocytes to die and have the remaining area turned into osteoblastic tissue.

The first step I believe was that the hypertrophic chondrocytes had something activated to cause them to secrete the chemical alkaline phosphatase (ALP). The waste that is expelled by the chondrocyte which had expression levels of ALP in it caused the area to become vascularized. Once the area becomes vascularized, then it become mineralized. Certainly mineralization is one of the key steps in this multi-step process. Then there are maybe 4-5 other steps after this, but we are not going to focus on those steps. In any series of chemical reactions, the easiest way to stop the series of reactions is to stop the first reaction from happening, thereby nipping the entire thing at the bud.

The idea that if we can just inhibit this one step, the mineralization step, means that the overall ECM still stay elastic and not bone hard. It may still go through the step of neo-vascularization become vascularized, and it may cause a run-away reaction of all the chondrocytes going through apoptosis but the matrix should stay in a cartilage-type tissue form for a much longer time. This would give the overall structure to possibly more time to expand longitudinally, thus making the bones longer than if they went their normal rate.

In conclusion we might consider the idea of also using Chondromodulin Type I or Type II, as well as GDF-5 in combination ,to possibly stop the vascularization as well.

If we stop both the mineralization and the vascularization, then we would stop the growth plate from ever closing, if we can figure out how to stop the hypertrophic chondrocytes from expression ALP through their waste.

{Tyler-Note that just because mineralization of the growth plates ceases does not mean that height will increase.  Stopping growth mineralization does not stop growth plate senescence see mice and rats.  Also, slowing down mineralization has been shown to decrease height in some instances.  It’s possible that it could increase height but experiments would be needed to test.  The better strategy is to stop growth plate senescence._

This is just some extra news that is worth showing the readers that the idea of taking a small piece of tissue from a patient, and then growing the cell into tissue in a lab culture in small microbiology petri dish, is very straight forward. This is something i already has been done at least once before by some other teams. Not only does the full tissue become synthesized in a culture dish, that tissue is reimplanted back into the cartilage defect areas of the patient. The entire process from the earliest step to the final step has been taken.

The last step now is to get the explanted tissue of chondrocytes to be grown into a columnar structure (via Thyroxine, refer to insanely critical study on power of Thyroxine to form growth plate organization ie columnar fashion back in 1994 by Dr. Ballock and Reddi Here) and have the released waste of proteoglycan and GAG (Glycoaminoglycan) into the ECM (Extracellular Matrix) to expand so that the tissue can expand, turning it into a “synthetic growth plate”. <– This step should not be that hard, and I believe it has already been accomplished in a research grant from 2012-2014.

Refer to the article “Doctors Have Discovered A Revolutionary Treatment For Knee Injuries” on Business Insider.

At Ohio State University, in the Wexner Medical Center, a  Dr. David C  Flanigan (His website is at www.flaniganmd.com) and his research team have been testing human cartilage grown in the lab. A patient named Taylor Landgraf, who was locally trying to get to the local gym and using a skateboard fell and tore the cartilage in his knee, as well as tearing his meniscus.

Taylor decided to look into getting some type of more modern type of treatment to repair his cartilage, since cartilage is probably one of the only tissue types which do not regenerate and heal itself, due to its unique structure. I would assume that he would get in contact with the Wexner Medical Center and somehow learn about the possibility of having lab grown tissue transplanted into his body.

So the researchers take a little bit of chondrocyte tissue as a type of tissue seed material from Taylor’s body. It is placed in a medium (agragose/hyaluronic acid/etc.) and grown in a cell line. The cartilage cells are replicated over and over again (I do have some issues here since it is well known that all cells have a limit to how many times they can replicated, similar to the idea of the Hayflick Limit).

Based on my own personal experience of listening to the speaker/CEO of RoosterBio, a company that sells mesenchymal stem cells, it was told to me that to have enough quantities of cells to form a reasonably large sized tissue, say even 2 cm by 2 cm, you would need around 60-200 Million cells. This suggests that if we assume cell mitosis, then to divide 10 times reveals a 2^10, or approximately 1000X magnification of cell numbers. What I am trying to say is that the amount of tissue you have to carve out of the patient may be quite sizable to have at least around 100,000 cells to start with (10^6). Assuming the Hayflick Limit of around 30 mitotic divisions (from the age of 20-30) , then we can start with much less. If we are assuming from the fetal stage, Wikipedia says instead that the limit of division is around 40-60 times.

Anyway, the result from starting with a cell line, and replicating it over and over again is a piece of living cartilage, about the size of a quarter (diameter of an inch, or 2.5 cm). You take that quarter sized cartilage, and carve it into the shape of the cartilage defect in the patient’s knee (or any other joint or location where cartilage has been scratched off). and pop it into the area where cartilage is missing.

So why is this worth mentioning? Is this big news or old news?

I wrote this post as a proof of concept. The type of cartilage that you get is most likely not going to be of the epiphyseal type, hyaline in nature. It will be fibrocartilage. The cartilage has an unorganized cartilage organization structure (ie. non-lamellar). Articular cartilage is hyaline in nature. Would the two different types of cartilage which now are next to each other bind at the boundaries and have something that will function overall at a reasonably good level? The reseatchers at this lab at OSU seem to think that this type of therapy is good enough for Taylor, at least semi-permanently for maybe 10-20 years. When that fibro-articular cartilage composite type starts to break down in 15 years, the researchers will have gone further on the tissue regenerative science and have something much better for him down the line in the future.

In fact, there is probably a much better technique for this Taylor patient which he should have tried, called Microfracture Surgery. Microfracture Surgery involves the surgeon just stabbing  the subchondral bone layer underneath the now grinded out articular layer of the knee epiphysis to make a hole. The stem cell type medium that exists in the cavity of the bones will leak out, and form as a type of blood clot turning into fibrocartilage tissue.

This way of doing it by the team with Flanigan seems a little too invasive, and not that necessary. However, it shows that researchers can grown cartilage in the lab from a patients own chondrocyte (or maybe even MSCs) and grown the cells into tissue, and reimplanted back into the body, and have that transplant to work just fine.

This is another step in the long process for what we ultimately want. It is a proof of concept for one of the most critical ideas and steps.