Something that has been really going through my thoughts recently is the possibility that the real limiting part of a possible non-invasive bone lengthening technique may not be the bones themselves, but the muscles and ligaments surrounding the bones.

This is the theory I propose the reader to think about.

Most ideas created to increase height works for the legs, and specifically the long bones in the legs. We know that the bones are surrounded by another main source of connective tissue, the muscles and ligaments. As for the ligaments, they are usually thin, strong connective tissue that are spread out in pattern. The muscles are instead bundled together to make them even stronger than the ligaments. We know from bodybuilding theory that even thought the muscles are strong, we can still tera the muscles and let them regrow and that is what causes muscle build up. In terms of elasticity, the muscles are obviously more elastic than the bones. From a common sense point of view, we say that the real reason we don’t get higher is because our bones are not elastic so we can’t stretch vertically up. And that is a reasonable arguement.

However, when we are talking about finding a non-invasive way to increase height, we find that often the reason the technique is so hard to achieve is because of the muscles covering the bone. To have any effect on the bones, we often have to apply some type of external stimuli through the muscle to the bone. And to do that besides a type of “action through a medium” technology (ie X.-Ray, radiation, ultrasounds, shockwaves, electrical fields), we almost always have to cut through the muscles using a solid That means that by definition, we are being non-invasive for almost all of our ideas.

What the LSJL method gets right is that you are doing something to the bone that is not really surrounded too much by muscle. Sure, there is the few layers of skin that cover the epiphysis but overal, the is not type of stimuli through the muscle to reach the bone.

I can promise to the readers right now that I can already devise 3 theretically sound probable ways to lengthen the actual long bones, however ,my ideas require that the covering tissues of the bone to be penetrated, lacerated, and distracted so that we can reach the bone and touch device or implant to the actual bone surface to have an effect. The main issue that makes it very difficult for me to devise a real non-invasive method is not the bone, but the surrounding tissue, the muscle, the cartilage, and the ligaments.

The problem then is that I am not a surgeon, much less orthopedic surgeon so I have no right to cut through human muscle to work on bone. Even if I propose a completely valid idea on how to lengthen the bone, there won’t be many orthopedic surgeons who would take my advice and be willing to integrate it into their practice.

In my research, there are many many ideas already out on how to lengthen bone, but only the bone. The muscle is often not considered, but when the actual technique has to be executed, then the people who have to do it, almost always a surgeon, have to consider the issue of dealing to muscle.

This post is one of the final studies which sort of culminates the type of research where we look at the ability modern science and technology have been able to come to find a way to repair, regenerate, and completely grow cartilage to be used for repair and implantation and transplantion using the gene therapy method.

Note: The study below is missing the references and citations, which is critical to possibly find other critical studies. I intentionally left out the really long list of references. Please use the link below to find what is needed.

Analysis & Interpretation

The study itself was to summarize the multiple ways medical researchers have tried to use gene therapy to heal articular cartilage defects. As noted from previous posts, the two main ideas was always to either inject the adenoviral vector with the right type of growth factors inside directly to the defect area or close to it, or to go the second path to first inject the vector into progenitor cells which will be altered to express more chondrogenic differentiation. The stem cells are then injected and implanted to the cartilage area. While the main issue had been that the cartilage that was formed was fibrocartilage, the noticing of hyaline cartilage makes the venture seem even more likely to have great effectiveness.

The researchers note that any damage to articular cartilage means that it becomes hard to repair. The articular cartilage has a strong resistance from becoming vascularized, which suggest that if a damage does occur, there is not blood vessels to transport the needed tissue repair elements needed to the right area. It seems that nutrients in the hyaline cartilage matrix might actually be diffused to the chondrocytes. When there is a partial defect, that area does not get healed from there being no way for the healing elements to reach the ara of the defect. When there is a more serious defect in the articular cartilage, where the fracture or defect goes deeper and actually makes a cut or puncture in the chondral bone in the subchondral layer, the  result is cartilage forming elements do come out of the inside of the bone but the cartilage they form is fibrocartilage, which has collagenous fibers that are not organized like hyaline cartilage.

When however not even the fibrocartilage is formed, that lead to pain for the subject so the medical professional might choose to stimulate the fibrocartilage formation process by doing a surgical technique known as abrasion anthroscopy, drilling, or microfracture surgery. The technique is reasonable effective and successful in removing the pain however it is well known that fibrocartilage is just not as good as the hyaline cartilage in the articular cartilage. There is much more Collagen Type 1 (while we are looking for Collagen type 2), there is more disorganization, etc. The result is that medical researchers have tried transplanting periosteum, perichondrium, and osteochondral parts to see what would happen .The short term effects is good in showing effectiveness in producing the hyaline cartilage they are looking for but the long term effects are not well known. It is suspected that one of the major problems is that there is not enough say periosteum or perichondrium available if a articular cartilage defect becomes too large.

The researchers seem to solve this problem by stating ,” Therefore, the autologous chondrocyte transplantation (ACT) procedure has been used clinically since 1987 in combination with a periosteal cover to treat chondral or osteochondral defects of the knee with good clinical results”

The top phrase means that the main way the researchers do it is to put the chondrocytes they get from an explant into a solid matrix (like a scaffold) and then implant the matrix which has the chondrocytes inside. Like one will learn from the Wikipedia article on the basic steps of Tissue Engineering, this is what tissue engineering is completely about. The result is that the symptoms in terms of pain does go away, at least for a short time while there is the autologous chondrocyte implant. However, again we see that the formation of really durable hyaline cartilage tissue is hard to form.

The whole point of the researchers can be summed up by their next sentence, “Therefore, tissue engineering approaches are being aggressively investigated in an effort to engineer cartilage in vitro to produce grafts to facilitate regeneration of articular cartilage in vivo“

The steps the researchers almost always goes through is…

1. The original chondrocyte cells or progenitor mesenchymal stem cells are taken from some enzymatic digestion process or an outgrowth culture.
2. The cells are put in a culture where they are slowly grown to increase in number until a cell colony (or sufficient cell number density) is formed.
3. The cells are then injected in a a scaffold or matrix because you need something to hold the chondrocytes in place for a 3-D space.
4. The matrix is then injected with growth factors to further grow the cell numbers and cartilage formation.
5. The matrix is then implanted into the cartilage defect.

The problems on why this general step by step procedure don’t seem to work well in new hyaline cartilage formation are…

• insufficient differentiation
• loss of transplanted cells or tissues
• matrix destruction
• integration failures

In the next section the researchers show that all of these problems which have been inhibiting the formation of new hyaline cartilage has been themselves been inhibited or have their inhibitory effects mostly stunted. There are now…

• morphogens and transcription factors that promote differentiation along chondrogenic lineages – thus solving the differentiation problem
• growth factors that promote matrix synthesis – thus solving the matrix destruction problem
• inhibitors of osteogenic or hypertrophic differentiation – it helps reduce the integration failures since it prevents osteogenic tissue from appearing, letting the chondrogenic tissues have a chance to form before being cut off by the bone making elements.
• antagonists that inhibit apoptosis – thus solving the loss of cells problem
• senescence or responses to catabolic cytokine – which solves the loss of tissues and cells problem

However even this new ideas and ways have their own set of problems. Those problems would be…

• delivery problems
• the limited half-life of many of the proteins that might be injected in vivo
• the difficulty in administration of the elements at a high enough concentration and for a long enough time for effectiveness
• the injected material can also affect non-targeted tissue and organs.

As a consequence, the researchers felt that it would be much more effective in use a gene therapy approach. The best approach would be to be able to alter the genetics of the chondrocytes close to the defect so that any type of hyaline cartilage regeneration will be more natural instead of trying to embed a transplant.

From Table 1 there will be a list of candidates to be put into a vector including all the types of TGF-Beta, IGF-1, BMPs, FGFs, and EGFs. The IHH, SHH, SMAD, and Sox genes are also named as potential regulators for chondrogenesis. Some of these components can be combined together to have a more effective regulatory role.

“There are many strategies that can be used to deliver exogenous cDNAs for the treatment of diseased or damaged cartilage….A key component for any gene therapy application is a vector that efficiently delivers the cDNA of interest to the target cell, and enables transgene expression of a suitable level and duration to affect the desired biological response. Furthermore, an understanding of the natural behavior of the target cell, such as its half-life, rate of division, and infectability with the vector are also essential to the effectiveness of the procedure.”

The basic principle of gene therapy is explained again. You put the cDNA into a vector, you inject the vector into a cell to change the way the DNA inside the cell to express genes in terms of types and amount. So far the ways gene therapy has been used in tissue engineering range from being the most complicated to being the simplest.

As we have said before, there is two main ways for vector use in intra-articular cartilage repair, the in vivo method and the ex vivo method. The in vivo method means putting the vector directly on the defect. The ex vivo method means putting the vector into cells first , and then transferring the cells to the defect. Which type of method to use depends on the gene being delivered and the vector to be used.

After this, the researchers would go into explain the 4 main ideas expressed in the diagram to the left.

They are …

Gene transfer to the synovium – add the vector to the synovium cells

Gene transfer to the cartilage defect – add the vector to the cartilage defect area for local trans-expression of the desired results from the chondrocytes in the area

Gene transfer to the chondrocytes – add to chondrocytes

Gene transfer to the mesenchymal stem cells – Note what the researchers said about the MSCs approach, “However, a successful use of MSCs to aid cartilage repair by means of generating a stable hyaline-rich cartilage repair tissue in vivo, likely requires the efficient delivery of factors to stimulate MSCs toward chondrogenesis, and maintenance of an articular cartilage phenotype without ossification, fibrinogenesis, or inflammation”

Implications For Height Increase

I think it is time to move our scope of analysis out more and see the forest and not the trees. We’ve gone really technical and deep into the details of this idea of gene therapy for intra-articular cartilage repair and we have to ask ourselves how we can use it to increase height. It has come to my awareness that the key to being able to say create a pseudo-growth plate after the natural growth plates close was to study the current medical science on how to regrow cartilage, but more specifically the hyaline cartilage. The best idea would be to create new epiphyseal cartilage, but the stacking nature of the chondrocytes to get the columns is something I still haven’t found much details on. There was only one study which showed that a chemical compound seems to result in the columnar arrangement of the chondrocytes. At this point I am just trying to study more on the way gene therapy works, on how tissue engineering works, and how the articular cartilage can possibly be regenerated after defects are formed. If this area of research is understood as a deep enough level then we can propose a way to using just a few injections get a part of the long bones to start creating hyaline cartilage. My theory is that as long as we can get hyaline cartilage (and maybe even fibrocartilage0 to be formed in a way that makes the tissue go completely around the cortical bone area, then we can use other methods like mechanical loading to get the bone to be lengthened with relative ease.

I had proposed many idea in the past on how to alter the bone and the cartilage in the long bone for increased length before using ideas like autologous chondrocyte transplantations, but the ideas were still only relatively vague and the details were missing. With this study I would be able to develop a more coherent and scientifically sound idea on how to use tissue engineering and gene therapy for cartilage regeneration for longitudinal increase in the long bones.

Note: The full study is copy and pasted below for reference if one desires to see the original paper.

Published in final edited form as:

Injury. 2008 April; 39(Suppl 1): S97–113.

doi:  10.1016/j.injury.2008.01.034

PMCID: PMC2714368

NIHMSID: NIHMS93905

Concepts in Gene Therapy for Cartilage Repair

Andre F. Steinert,¹ Ulrich Nöth,¹ and Rocky S. Tuan²

Summary

Once articular cartilage is injured, it has a very limited capacity for self-repair. Although current surgical therapeutic procedures to cartilage repair are clinically useful, they cannot restore a normal articular surface. Current research offers a growing number of bioactive reagents, including proteins and nucleic acids, that may be used to augment different aspects of the repair process. As these agents are difficult to administer effectively, gene transfer approaches are being developed to provide their sustained synthesis at sites of repair.

To augment regeneration of articular cartilage, therapeutic genes can be delivered to the synovium, or directly to the cartilage lesion. Gene delivery to the cells of the synovial lining is generally considered more suitable for chondroprotective approaches, based on the expression of anti-inflammatory mediators. Gene transfer targeted to cartilage defects can be achieved by either direct vector administration to cells located at or surrounding the defects, or by transplantation of genetically modified chondrogenic cells into the defect. Several studies have shown that exogenous cDNAs encoding growth factors can be delivered locally to sites of cartilage damage, where they are expressed at therapeutically relevant levels. Furthermore, data is beginning to emerge indicating, that efficient delivery and expression of these genes is capable of influencing a repair response toward the synthesis of a more hyaline cartilage repair tissue in vivo. This review presents the current status of gene therapy for cartilage healing and highlights some of the remaining challenges.

Introduction

The application of gene transfer to articular tissues was pioneered by Evans and co-workers, as a means to treat arthritis [46,49]. Initial encouraging experiments in animal models using retroviral-mediated gene delivery formed the basis for a clinical trial to evaluate the safety and feasibility of using gene therapy for rheumatoid arthritis [46,49,59-61,148]. The study was completed without incident; the procedure was well-tolerated by the nine participants, and intra-articular gene transfer and expression was observed in all joints treated [46,49]. The relative success of these studies suggests that this technology may have application in treating a number of articular disorders for which current treatment modalities are unsatisfactory. Compared to the treatment of chronic or genetic diseases, where likely a lifelong expression of a corrective transgene is required, the use of gene transfer techniques to facilitate musculoskeletal tissue repair offers perhaps an immediate opportunity for a clinical application of gene therapy, as it may only require transient, localized expression of a specific transgene product. Whereas good success has been achieved by gene transfer to bone healing [9], augmenting the repair of focal articular cartilage defects by gene transfer has not been straightforward. Current research indicates that the design of a successful genetic approach for cartilage repair includes a refined strategy of gene delivery that meets the complexities of treating this tissue. This review aims to summarize some of the basic principles of cartilage injury and regeneration, and comments on the pros and cons of recent gene therapy approaches to repair, as well as future challenges.

Cartilage injury and limitations of current treatments

Hyaline articular cartilage is a highly specialized tissue that protects the bones of diarthrodial joints from forces associated with load bearing, friction and impact. Although a remarkably durable tissue, once articular cartilage is injured, it has very limited capacities for self-repair. In partial thickness defects, where a lesion is wholly contained within the articular cartilage, there is no involvement of the vasculature. Consequently, chondroprogenitor cells in blood and marrow cannot enter the damaged region to influence or contribute to the reparative process. Resident articular chondrocytes do not migrate to the lesion, and production of a reparative matrix by these cells does not occur. As such, the defect is not filled or repaired and essentially remains permanently [21,80]. Full thickness cartilage injuries result in damage to the chondral layer and subchondral bone plate, causing rupture of blood vessels, and hematoma formation at the injury site. In this case, a repair response is initiated that results in the formation of a fibrocartilage repair tissue within weeks [21,80].

In focal cartilage defects, where a stable fibrocartilaginous repair tissue has not formed, and patients are suffering clinical symptoms such as pain and swelling, surgeons aim to promote a natural fibrocartilaginous response, by using marrow stimulating techniques, such as abrasion arthroplasty, Pridie drilling, or microfracture. These procedures are cost effective and clinically useful, as patients often have reduced pain and improved joint function, and are therefore generally considered as first-line treatment for focal cartilage defects [22,121,122,161,162]. However, fibrocartilage has inferior mechanical and biochemical characteristics compared to normal hyaline articular cartilage. It is poorly organized, contains significant amounts of collagen type I, and is susceptible to injury. The inferior repair matrix breaks down with time and loading, which ultimately leads to premature OA [21,80]. Therefore, as outlined in other articles of this issue, the aim of modern therapeutic techniques is to achieve a more hyaline-like cartilage repair tissue by transplanting tissues or cells. Tissue transplantation procedures such as periosteum, perichondrium, or osteochondral grafts have shown positive short term results for a number of patients, but the long term clinical results are uncertain, with tissue availability for transplant being a major limitation, especially in large cartilage defects [19,22,23,71,80]. Therefore, the autologous chondrocyte transplantation (ACT) procedure has been used clinically since 1987 in combination with a periosteal cover to treat chondral or osteochondral defects of the knee with good clinical results [20,121,144,145]. Modern modifications of this procedure include embedding chondrocytes in a three dimensional matrix before transplantation into cartilage defects [15,114,123]. Despite these advances, most surgical interventions only result in improvement of clinical symptoms, such as pain relief, but none of the current treatment options has regenerated long-lasting hyaline cartilage repair tissue yet [22,80,121,144]. Therefore, tissue engineering approaches are being aggressively investigated in an effort to engineer cartilage in vitro to produce grafts to facilitate regeneration of articular cartilage in vivo. In most cases, cells are harvested by enzymatic digestion or outgrowth culture, which are thereafter extensively expanded in culture. The cells are then seeded onto various biologically compatible scaffolds, and cultured in the presence of a specific cytokine or growth factor, or a cocktail of bioactive factors. However, despite promising in vitro data with several approaches, a significant improvement compared to current cartilage repair modalities, has yet to be achieved. Many challenges thus remain for successful cell-based cartilage repair approaches to form hyaline repair tissue in vivo [23,80,92,177]. Impairments of hyaline neo-cartilage formation is likely due to a number of reasons, including insufficient differentiation, loss of transplanted cells or tissues, matrix destruction and integration failures, which all can occur due to various reasons.

Candidate gene products

In recent years, several factors have been identified that might be functional in augmenting different aspects of cartilage tissue repair. Of particular interest are morphogens and transcription factors that promote differentiation along chondrogenic lineages, growth factors that promote matrix synthesis, inhibitors of osteogenic or hypertrophic differentiation, antagonists that inhibit apoptosis, senescence or responses to catabolic cytokines (Table 1). Several of these substances have shown promise in animal models of cartilage repair and regeneration, but their clinical application is hindered by delivery problems [103,164,171]. Due to the limited half-lives of many proteins in vivo, they are particularly difficult to administer to sites of cartilage damage at therapeutic concentrations and for sustained periods of time. Localized delivery of these agents without involvement of non-target organs has also proven to be problematic [164,171]. We suggest that these limitations may be overcome by adapting appropriate gene transfer technologies. In particular, it should be possible to develop techniques for transferring therapeutic genes encoding the necessary gene products to cells at the sites of injury or disease, for sustained local expression at high levels with minimal collateral exposure of non-target tissues [164,171]. In this manner, the proteins of interest are synthesized locally by cells and are presented to the microenvironment in a natural fashion. Furthermore, recombinant proteins produced by overexpression in bacteria may have altered activity, since they may not be similarly modified post-translationally as when synthesized by a mammalian cell [113].

Table 1

Classes of gene products that aid cartilage repair

The list of potentially useful cDNAs for cartilage repair (Table 1) comprises members of the transforming growth factor (TGF)-β superfamily, including TGF-βs 1, 2, and 3, several of the bone morphogenetic proteins (BMPs), insulin-like growth factor (IGF)-1, fibroblast growth factors (FGFs), and epidermal growth factor (EGF), among others (reviewed in [74,103]). Other secreted proteins, such as indian hedgehog (IHH) or sonic hedgehog (SHH), play key roles in regulating chondrocyte hypertrophy [185], and may also prove to be beneficial for modulating the chondrocytic phenotype of grafted cells. Another class of biologics that may be useful in cartilage repair is transcription factors that promote chondrogenesis or the maintenance of the chondrocyte phenotype. SOX9 and related transcription factors like L-SOX5, and SOX6 have been identified as essential for chondrocyte differentiation and cartilage formation [98]. Signal transduction molecules, such as SMADs, are also known to be important regulators of chondrogenesis [76]. As these molecules function completely intracellularly, they cannot be delivered in soluble form, and gene transfer might be the only way to harness these factors for repair. Alternatively, delivery and expression of cDNAs encoding specific extracellular matrix (ECM) components such as collagen type II, tenascin, or cartilage oligomeric matrix protein (COMP), may also be used to support production and maintenance of the proper hyaline cartilage matrix [37].

Prevention or treatment of cartilage loss may also require the inhibition of the actions of certain pro-inflammatory cytokines, such as interleukin (IL)-1 and tumor necrosis factor (TNF)-α, as these are important mediators of cartilage matrix degradation and apoptosis after trauma and disease. Therefore, anti-inflammatory or immunmodulatory mediators, such as interleukin-1 receptor antagonist (IL-1Ra), soluble receptors for TNF (sTNFR) or IL-1 (sIL-1R), IL-4 or IL-10, inhibitors of matrix metalloproteinases, and others may be administered to effectively reduce loss of repair cells and matrix [148].

Inhibitors of apoptosis or senescence, such as Bcl-2, Bcl-XL, hTERT, i(NOS) and others (Table 1), may also be beneficially employed in order to maintain cell populations at the injury site, which are capable of favorable repair responses [39,41]. Different candidate cDNAs might also be administered in combination, especially when favoring complementary therapeutic responses. For example, the combined administration of an anabolic growth factor (e.g. IGF-1) together with an inhibitor of the catabolic action of inflammatory cytokines (e.g. IL-1Ra) has the potential to control matrix degradation as well as to allow partial restoration of the damaged cartilage matrix [73,132].

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Strategies to gene therapy in the repair of articular cartilage

There are many strategies that can be used to deliver exogenous cDNAs for the treatment of diseased or damaged cartilage. For a successful approach, several factors have to be taken into account, including the extent of cartilage pathology, disease processes, and the biological activity of the gene product, among others. A key component for any gene therapy application is a vector that efficiently delivers the cDNA of interest to the target cell, and enables transgene expression of a suitable level and duration to affect the desired biological response. Furthermore, an understanding of the natural behavior of the target cell, such as its half-life, rate of division, and infectability with the vector are also essential to the effectiveness of the procedure. The properties of commonly used vectors in gene therapy applications are summarized in Table 2, and have been extensively reviewed elsewhere [136,168]. Gene-transfer strategies in which these vectors are currently used for cartilage repair, range from those as simple as direct delivery of a vector to a defect, to synthesis of cartilaginous constructs through genetically augmented tissue engineering procedures. We will present below an overview on the properties of commonly used vectors in gene therapy applications (Table 2), and will discuss their use in the context of the different delivery strategies to cartilage defects.

Table 2

Nonviral and viral vectors for orthopaedic gene therapy applications

There are two general modes of intra-articular gene delivery, a direct in vivo, and an indirect ex vivoapproach (Figure 1). The direct in vivo approach involves the application of the vector directly into the joint space, whereas the ex vivo approach involves the genetic modification of cells outside the body, followed by re-transplantation of the modified cells into the body. The choice of which gene transfer method to use is based upon a number of considerations, including the gene to be delivered, and the vector used. In general, adenovirus, herpes simplex virus, adeno-associated virus vectors, lentivirus and non-viral vectors may be used for in vivo and ex vivo delivery (Figure 1, Table 2). Retroviral vectors, because of their inability to infect non-dividing cells, are more suited for ex vivo use. Ex vivo approaches are generally more invasive, expensive and technically tedious. However, they permit control of the transduced cells and safety testing prior to transplantation. In vivo approaches are simpler, cheaper, and less invasive, but viruses are introduced directly into the body, which limits safety testing.

Figure 1

Gene transfer approaches for the treatment of cartilage defects. (A) For in vivo gene transfer, free vector is either injected directly into the joint space, or incorporated into a biologically compatible matrix before implantation into a cartilage defect …

Toward the treatment of damaged articular cartilage, the three primary candidate cell types to target genetic modification are synovial lining cells, chondrocytes, and mesenchymal stem cells.

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Gene delivery to the synovium

The simplest strategy for gene delivery to diseased joints is by direct intra-articular injection of a recombinant vector [60,61]. For this application, the two primary tissues to consider are cartilage and synovium. Within articular cartilage, chondrocytes are present at low density and reside at varying depths within the dense matrix. Because of this, efficient genetic modification of chondrocytes in situhas not been effectively achievable [32,62,170,192]. The synovium, in contrast, is a tissue that is much more amenable to gene delivery. It usually exists as a thin lining of cells that covers all internal surfaces of the joint except that of cartilage, and thus has a relatively large surface area, and is therefore the predominant site of vector interaction. Direct intra-articular injection of vector or modified cells results in synthesis and release of therapeutic proteins into the joint space, which then bathe all available tissues, including cartilage. Using various types of vectors in ex vivo and in vivo approaches, considerable progress has been made towards defining the parameters critical to effective gene transfer to synovium and prolonged intra-articular expression. The effectiveness of synovial gene transfer of various transgenes is well documented in research directed towards rheumatoid arthritis [148]. Ex vivogene delivery to joints has since been taken into phase I clinical trial and shown to be feasible and safe in humans with RA [46,50].

Although most of the work involving direct intra-articular gene delivery has been focused toward the study and treatment of RA, data are beginning to emerge of its potential for treating OA (reviewed in [47]), and to augment repair approaches of focal cartilage defects (Table 3) [31,58,164,171]. For example, encouraging results have been reported for adenovirally delivered IGF-1 or IL-1Ra using animal models for OA and localized cartilage injury [32,54]. While it is possible to achieve biologically relevant levels of transgene expression by both direct and ex vivo gene transfer to synovium, this approach is not compatible with the delivery of certain growth factors. For example, adenoviral mediated delivery of TGF-β1 or BMP-2 to the synovial lining was found to generate joint fibrosis, extreme swelling, osteophytes and cartilage degeneration [8,56,57,120]. Considering these results in the context of cartilage repair, synovial gene transfer may be more suitable for delivering chondroprotective agents rather than strong anabolic transgenes with pleiotropic effects of their products. Many anti-inflammatory cytokines have this property (see Table 1).

Table 3

Therapeutic gene transfer studies to repair focal articular cartilage defects

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Gene delivery to cartilage defects

For the gene-based delivery of certain growth factors or intracellular proteins, a strategy whereby the transgenes are more localized, and the gene products contained within the cartilage lesion, appears to be most prudent. Possibly, the most direct manner by which to achieve this goal is by implantation of a three-dimensional matrix pre-loaded with a gene delivery vehicle into a defect, allowing infiltrating cells to acquire the vector and locally secrete the stimulating transgene products [18,48]. Genetically activated implants have been designed to augment the healing of bones, ligaments and also cartilage [17,34,48,140,141,150]. For example, hydrated collagen-glycosaminoglycan matrices containing adenoviral vectors have been found to promote localized reporter gene expression in vivo, following implantation into osteochondral defects in rabbit knees, for at least 21 days [140]. However, given the usually limited cell supply at the cartilage lesion site, it is not yet known whether this type of approach can induce a sufficient biological response for repair. In order to increase the cellularity of the graft, while preserving the feasibility of the procedure within one operative setting, the genetically activated matrix could be mixed together with autologous cells, which are intraoperatively readily available, e.g. cells from bone marrow aspirates (Figure 1). Such an abbreviated, genetically enhanced tissue engineering approach would thus save time and costs, while avoiding labor-intensive ex vivo culture of cells [48,140]. Their limitation, however, is the lack of control over gene transfer following implantation.

As there are several advantages, gene transfer has mostly been used to augment ex vivo cell delivery approaches for cartilage repair (Figure 1). Such an approach delivers a pure population of cells, that can be selected under controlled conditions; the graft is highly cellular, localizes transgene expression to the injury site without administration of free vector, and there is the possibility for safety testing prior to transplantation. In the context of ex vivo gene delivery to cartilage defects, several experimental studies have been performed, exploring gene transfer to chondrocytes or mesenchymal progenitor cells.

Gene transfer to chondrocytes

A major advantage of using autologous chondrocytes as cell source for cartilage repair is that their application has already found the way out of the experimental stadium to clinical practice [20]. In recent years, autologous chondrocyte transplantation (ACT) has become a clinically adopted procedure for cartilage defects, especially when marrow stimulation techniques failed to generate good clinical results [145]. In order to further improve the quality of the repair tissue, attempts have been made to enhance this procedure by the use of genetically-modified chondrocytes. Although chondrocytes have been somewhat resistant to transfection with plasmid DNA, certain lipid-based formulations have been found to enhance the efficiency of DNA uptake [106]. Viral based vectors, however, are capable of generating far higher levels of transgene expression with greater persistence. Monolayer expanded chondrocytes are readily transduced by viral vectors, such as Moloney Murine Leukemia Virus (MLV), lentivirus, adenovirus and AAV. Adenoviral-mediated delivery of various transgenes, such as TGF-β1, BMP-2 , IGF-1 or BMP-7, has been shown to stimulate the production a cartilage-specific matrix rich in collagen type II and proteoglycans, and a decreased tendency towards dedifferentiation [75,130,131,157,159]. Transfer of cDNA encoding matrix molecules, such as the collagen type II minigene, led to enhanced extracellular matrix production of human fetal chondrocytes [37]. Transduction with the transcription factor SOX-9 increased collagen type II expression of chondrocytes in three-dimensional culture in vitro [99,167], whereas overexpression of the transcription factor Runx-2 (Cbfa-1) stimulated chondrocyte maturation and induced a hypertrophic phenotype, expressing high levels of collagen types II and X, alkaline phosphatase and osteogenic marker genes [44,84].

Having shown that chondrocyte biology can be positively influenced by genetic modification, research focus has shifted towards their efficient delivery to cartilage lesions. The first approach would be the delivery of genetically modified chondrocytes in suspension. Several studies have shown that genetically modified chondrocytes are capable of expressing transgene products at functional levels following engraftment onto cartilage explants in vitro [42]. In such systems, genetic-modification with IGF-1 [107], FGF-2 [109], or SOX9 [33] led to significant resurfacing and thicker tissue enriched with proteoglycans and collagen type II, compared to transplanted control cells [106]. In addition, adenoviral-mediated IL-1Ra gene transfer to chondrocytes resulted in resistance to IL-1-induced proteoglycan degradation after engraftment [11]. As an alternative to delivery in suspension, efforts have also been made to augment tissue engineering procedures using genetically modified chondrocytes (Figure 1C). For this, the cells are transduced/transfected in monolayer and then seeded into a matrix for subsequent transplantation into chondral or osteochondral defects. In such three-dimensional culture systems, several transgenes have shown promising results in maintaining and promoting the chondrogenic phenotype in vitro, including TGF-β1, BMP-2, -4, -7, IGF-1, SOX9 among others [164,171,177].

Initial studies demonstrated that chondrocytes efficiently expressed reporter genes in chondral and osteochondral defects following genetic modifications with adenoviral, AAV, retroviral or plasmid vectors, and that transgene expression was prolonged for several weeks when the genetically-modified chondrocytes were seeded in three-dimensional matrices [12,82,89,108]. Results of efficacy studies are just beginning to emerge, showing the effects of genetically modified chondrocytes in cartilage lesions in vivo (Table 3). In an ex vivo approach, adenovirally-transduced chondrocytes expressing BMP-7 [75], incorporated in a matrix of autogenous fibrin, were implanted into full thickness articular cartilage defects in horses [75]. Four weeks after surgery, an increased tissue volume and accelerated formation of a proteoglycan and collagen type II rich matrix could be observed in the BMP-7 treated defects compared to control defects treated with irrelevant marker genes. However, after 8 months, the levels of collagen type II and proteoglycan, and the mechanical characteristics of the treated defects compared to the controls were similar. This was attributed in part to the declining number of allografted chondrocytes that persisted in the defects after 8 months [75]. Nevertheless, it is encouraging that genetically modified chondrocytes can be used to augment a cartilage repair process in a large animal model.

Gene transfer to mesenchymal stem cells

The use of autologous chondrocytes for the repair of articular cartilage is limited, as they have to be isolated from a very limited supply of healthy non-weight-bearing articular cartilage, which has to be surgically removed, with the risk of donor site morbidity. Furthermore, chondrocytes dedifferentiate during expansion with a subsequent loss of the chondrocytic phenotype. With regard to cell- and gene-based approaches to cartilage repair, mesenchymal progenitor cells, also referred to as mesenchymal stem cells (MSCs), provide an attractive alternative to chondrocytes. Although no clear phenotype has been described, through the use of the proper culture conditions, expanded MSCs can be stimulated to differentiate along specific pathways such as chondrogenesis, adipogenesis, and osteogenesis [23,25,26,28,86,92,134,135,146,175-177]. MSCs have been isolated from several sources, including bone marrow [147], trabecular bone chips [134], adipose tissue [198], periosteum, perichondrium and others, and have been shown to maintain their multilineage potential with passage in culture [195]. In order to harness MSCs for cartilage tissue engineering, analyses of the appropriate three-dimensional microenvironment to stimulate MSCs toward chondrogenesis in vitro und in vivo have been performed extensively, with factors such as TGF-β1, 2, -3, and BMP-2 emerging among the most popular candidates (see also Table 1). This research has led to the first clinical application of autologous bone marrow stromal cells for the repair of full-thickness articular cartilage defects in humans, which resulted in stable fibrocartilage tissue formation at the defect site [93,187]. However, a successful use of MSCs to aid cartilage repair by means of generating a stable hyaline-rich cartilage repair tissue in vivo, likely requires the efficient delivery of factors to stimulate MSCs toward chondrogenesis, and maintenance of an articular cartilage phenotype without ossification, fibrinogenesis, or inflammation [23,80,177].

In order to meet these requirements, gene therapy approaches hold promise for efficient implementation in cartilage repair procedures. In this context, MSCs are readily transduced by recombinant adenoviral, retroviral, lentiviral, AAV [24,57,195,196] and foamy viral vectors (A. Steinert and A. Rethwilm, unpublished observation). Specific liposomal formulations were used with some efficiency [69,106,107], as well as molecular vibration-based methods [160]. In vitro chondrogenesis has been shown, following plasmid-mediated BMP-2 and BMP-4 [1,163], retrovirus-mediated BMP-2 [27], and adenovirus-mediated BMP-13 gene transfer in the murine mesenchymal progenitor cell line C3H10T1/2. Marrow-derived, primary mesenchymal progenitor cells, genetically modified to express TGF-β1 or BMP-2, were also found to undergo chondrogenesis in aggregate culture, in contrast to IGF-1 modified cultures and reporter gene controls [196]. Interestingly, chondrogenesis in these cultures was also dependent on the level and duration of transgene expression and the viral load, indicating that these factors have to be carefully optimized for a successful in vivo translation of this technology [196].

Some first studies have been performed applying MSC-mediated gene delivery for cartilage repair in vivo. A variety of reporter genes have been successfully delivered to osteochondral defects via periosteal, perichondral or marrow derived MSCs [116,138,140,164,171]. Only a few studies have been conducted using therapeutic genes via MSCs thus far.

A genetically enhanced tissue engineering approach used constructs fabricated of retrovirally-transduced periosteal cells expressing BMP-7, which were seeded into polyglycolic acid scaffolds before transplantation into rabbit osteochondral defects [116,117]. The defects treated with BMP-7 modified progenitors revealed improved regeneration tissue of cartilage and bone, compared to controls after a maximum of 12 weeks post-implantation. In a study using a similar experimental approach, genetically modified periosteal cells transduced to express sonic hedgehog (SHH) were compared to the delivery of the BMP-7 cDNA, which resulted in a better overall repair of the SHH compared with the BMP-7 treated defects after 12 weeks postoperatively, and both were superior to marker gene controls [67]. Using the same animal model, constructs of a collagen type I hydrogel and marrow derived MSCs following liposomal GDF-5 (CDMP-1) gene delivery were shown to enhance cartilage repair compared with marker gene controls [90].

Another approach to study gene-induced chondrogenesis in vivo was devised by Gelse et al. who used gene transfer to MSCs for the repair of partial thickness cartilage lesions in rats [57]. The MSCs were isolated from rib perichondrium and, following adenoviral-modification with Ad.BMP-2 and Ad.IGF-1, delierved via a fibrin glue matrix to partial thickness cartilage lesions of the patellar groove. Both treatment with BMP-2 and with IGF-1 resulted in formation of improved repair tissue rich in collagen type II and proteoglycans, compared with the naïve and Ad.LacZ controls after 8 weeks [57]. However, the majority of BMP-2 treated joints showed signs of ectopic bone formation and osteophytes, which were not present in the knees of the IGF-1 treated defects [57].

In order to simplify elaborate and expensive ex vivo tissue engineering procedures, efforts are underway to facilitate gene delivery approaches to stimulate MSCs at the defect site in vivo toward chondrogenesis. The simplest way of achieving this aim is maybe via direct vector delivery to the cartilage defect site. Toward this end, direct application of recombinant AAV vectors in suspension [32], or of adenoviral vectors incorporated in hydrated collagen-glycosaminoglycan matrices [140] have been found to promote localized transgene expression within the repair tissue formed, following transplantation into cartilage lesions in vivo. However, considerable vector leakage to adjacent synovium was observed [140]. In an attempt to augment this kind of approach with an autologous cellular and space-filling entity, Pascher and colleagues demonstrated that when fresh bone marrow aspirates were mixed with a solution of recombinant adenoviral vectors and allowed to coagulate, MSCs within the coagulum acquired and expressed the transgene for several weeks after implantation into osteochondral defects in rabbits [140]. Studies are underway to investigate how these advances can be harnessed to achieve cartilage repair.

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Challenges for gene therapy to promote cartilage repair

Currently used cartilage repair approaches, both experimental and clinical, are still far from generating a repair tissue that is comparable to the native cartilage tissue quality and stability. To tackle various obstacles toward successful repair, including matrix degradation, differentiation or integration insufficiencies, or loss of the transplanted cells and tissues, efficient delivery of chondrogenic, anti-inflammatory, and anti-oxidative factors seems to be crucial (Table 1). As most of these factors are recombinant proteins, which have short half lives, a repeated local administration is likely to be necessary to achieve the desired result, thus presenting delivery problems. Gene transfer techniques might be adopted that could overcome the limitations of the current treatments for damaged articular cartilage. The current concepts in gene therapy for cartilage repair are reviewed here. Various approaches have been shown to be suited for efficient transfer of exogenous cDNAs to cartilage defectsin vivo, and for achieving sustained expression of the corresponding gene products. Initial efficacy studies indicate that gene-transfer techniques are potent tools that can indeed stimulate a relevant biological response in vivo (Table 3). To date most approaches delivered a strong anabolic transgene aiming to achieve formation of a hyaline-like cartilage repair tissue in vivo, but with limited long-term success thus far. As more data surfaces, a clearer picture of the functional boundaries of current approaches appears. The future challenge therefore is to determine which combination of transgenes will be most suitable for which aspects of repair, and how best to deliver and express them.

Toward this end, the use of more refined vector systems seems to be crucial. Current gene transfer studies to cartilage repair have used vector systems with strong, viral-based promoters enabling very high levels of expression, thus facilitating study of the biological effects that may be achieved with a particular gene and gene delivery method. However it is likely that the stimulation of faithful synthesis of the complex architecture of articular cartilage, followed by its maintenance long-term will require the use of more sophisticated vector systems capable of coordinate control of expression. As many gene products proposed for use can have detrimental side effects if overexpressed in non-target organs such as the heart, lung or kidney, the characterization of the duration of expression in vivo and the biodistribution of vector and/or genetically modified cells following delivery, will be critical. Toward this end, there are several types of cartilage-specific regulatory elements that have been characterized and that might be incorporated into gene delivery systems, such as promoters for the cartilage-derived retinoic acid-sensitive protein (CD-RAP), the procollagen type II α1 (COL2A1), or the aggrecan gene [96,100,127,128,158,173,180,191,197].

Because cartilage injuries are not life-threatening, the safety of gene transfer approaches for repair is of particular importance. To harness the potential of this technology for clinical use is therefore strongly dependent on the use of safe and efficient vectors, transgenes and delivery systems.

This post is more of an extension of the first article “New Proposed Height Increase Method Using Gene Therapy On Engineered Pluripotent Mesenchymal Cells (Breakthrough!)“. It gives the method a more solid theoretical foundation and validity. In the first study, the researchers used recombinant adenoviral vectors carrying the human BMP-2 gene (rh-BMP2) to transduce MSCs to have a prolonged effect of increasing the expression of the protein which the gene is supposed to make.

We saw that bone formation and growth can be regulated or increased from increasing the expression of certain types of growth factors by using an implanted vector.

Update 2/16/2013: At this point I would have to say that idea I had propose in the previous post above has some theoretical backing behind it but is also quite flawed once I go back and read the studies and see what they are actually implying. Those studies showed that the growth factors being made by the mesenchymal stem cells, caused osteogenic effects, not chondrogenic. This just goes to show how science and research and better understand causes out previous understanding to be proven wrong and for us to constantly be changing our level of understanding of the science.

Analysis & Interpretation

Let’s first look at the first study. We are learning a lot from this study. It seems that the BMP-2 which we are so familiar with seem to cause bone cells like osteocytes and osteoblast than say cartilage cells if you inject the growth factor straight into say the epiphysis of long bones in a lab rabbit. The bone formation can be seen in vitro but also in vivo however for the BMP-2 to work in vivo (in the actual lab animal) it has to have really high concentrations, around the mg level of concentration instead of the micrograms which most growth factors are found in. Plus, the effects of the growth factor is really fast and short in time duration. There is an initial injection of the growth factor but that will only get diffused and degenerated after say a few days. To get around the issue of only a small dose and short time frame of dosage, the idea was to inject cells into the rabbit leg bone defects than growth factors.

The cells themselves are the Mesenchymal Stem Cells (MSCs) which can be found from a bone marrow removal and then centrifuged or have some process done to them which would cause the MSCs to be separated, filtered, and then purified. These marrow derived MSCs would be place in a culture. The vectors known as recombinaant adenoviral vector will have the human BMP-2 gene (Adv-BMP2) inside oft  them already. The vectors are added into the culture. The cell membrane of the MSCs which are pluripotent area willing to let the vectors get hooked to a receptor of the outer cell membrane and either let the entire vector inside or just let the gene which the vector was housing to be popped inside into the intercellular matrix of the cell. I would guess then that gene finds it’s way to the surface of the nucleus membrane, get through that, and then somehow cause the genome and chromosomes of the nucleus to start turning on or turning off the specific genes for the specified growth factor. For this case, it is the BMP-2. The cells start expressing the BMP-2 and they start turning into aka differentiating into the bone cell type aka osteoprogenitor cells. The researchers can confirm that the MSCs are turning into bone cells like osteocytes/osteoblasts by using assays and histology to check the level of alkaline phosphatase expression, the amount of Collagen Type I, another compound called osteopontin, osteocalcin, and matrix mineralization.

At the microscopic level with say one cell or a culture of same type pluripotent MSCs this entire changing of cell type is possible using elementary gene therapy techniques.

Now let’s look at the 2nd study. Note that the 1st and 2nd studies/articles are written by the same group of university researchers so the two articles are essentially the same. Right off the bat the researchers state this point, “Bone marrow-derived mesenchymal stem cells are pluripotential cells that have the capacity to differentiate into an osteoprogenitor line”. This was proved from the 1st study. MSCs you find from the bone marrow can indeed differentiate into the bone cell type. They conclude by saying, “We conclude that it is possible to successfully transduce mesenchymal stem cells with the gene for BMP-2 such that these cells will produce the BMP-2 protein in vitro. Further, the transduction results in transformation of these cells into an osteoprogenitor line capable of producing bone in vivo. These data suggest the feasibility of employing gene therapy using recombinant adenoviral vectors as a tool for enhancing spine fusion. Further work to improve the fidelity and longevity of the gene transfer is warranted.” In both articles the researchers put the cells with the vectors for expression BMP-2 into the rabbit’s intervertebral region, between the lumbar vertebrate #5 and #6.

Now let’s look at the 3rd study. Again the researchers are trying to prove that the MSCs you get from bone marrow can turn themselves aka differentiate into the bone type of cells. The setup is however a little different.

• First, there is a culture but there is two types of cultures the MSCs will be in to test for differentiation capacity and extent, a monolayer and then another three-dimensional alginate culture systems
• Second, the MSCs will be tested with getting the adenoviral vectors having both BMP-2 encoding genes and BMP-6 encoding genes.

The results show that the BMP-2 encoded genes seem to work better in getting the pluripotent MSCs to differentiate in both of the culture types for better and/or faster bone formation.

The Actual Method

In the previous post I had talked about the idea of using gene therapy to cause height increase. That was a two step process,

• First is the injection of pluripotent MSCs which would only express chondrogenic genes causing it to produce only chondrogenic growth factors. – This would hopefully result in all the stem cells being injected to turn into chondrocytes.

• Second is the injection of already differentiated chondrocytes with their nucleus getting the adenoviral vector for the up-regulated expression thus the production of growth factors which will cause the chondrocytes , both already inside the epiphysis and then next chondrocyte group injection, to come together and proliferate thus forming a new cartilage inside the epiphysis.

The idea for the previous post was a theory and a proof of concept on how to use the gene therapy techniques we know right now to help us increase height. The truth is that the elements to do the entire process is the basic process of tissue engineering in general. there is always these steps

1. You take a sample from the person’s body which has some stem cells
2. You put the sample through some type of machine to filter the stem cells
3. The stem cells are put in a culture of a certain type to grow them to a certain number.
4. You put the stem cells in some type of matrix or porous scaffold.
5. You add growth factors to the stem cells to make it grow to proliferate
6. You implant that porous scaffold into the human which they came from.
7. You watch to see how well the human body accepts the scaffold filled with the stem cells.

This is tissue engineering. You already have many biomedical researchers doing this type of research for finding ways to regenerate or heal cartilage, but more specifically the articular cartilage in the major joints like the knees and hips.

This step in general is something which I have already done enough research to understand the basic steps of tissue engineering.

Now we are learning how to incorporate genetic engineering/ gene therapy with the tissue engineering to make the stem cells we put in either or both the scaffold or culture to make them do certain things like differentiate, proliferate, hypertrophy, release certain types of growth factors.

For the new and better improve method for height increase is to focus only on the step where the cells themselves have something done to them more than just putting growth factors with them in a culture or scaffold. It is to put a vector with a certain type of protein producing gene in them. This means that the effect for growth factor production or prolfieration will be increased. The dosage will be go along for a longer amount of time meaning that the desired process is increased. Instead of just the first dosage of growth factors effecting the stem cells, the vector will cause the nucleus of the cells themselves to go through the process intrinsically and make it more permanent, at least until the cells go through apoptosis.

From PubMed study 1 In vitro and in vivo induction of bone formation using a recombinant adenoviral vector carrying the human BMP-2 gene.

Calcif Tissue Int. 2001 Feb;68(2):87-94. – Cheng SL, Lou J, Wright NM, Lai CF, Avioli LV, Riew KD.

From PubMed study Induction of bone formation using a recombinant adenoviral vector carrying the human BMP-2 gene in a rabbit spinal fusion model.

Calcif Tissue Int. 1998 Oct;63(4):357-60. – Riew KD, Wright NM, Cheng S, Avioli LV, Lou J.

Source – Department of Orthopaedic Surgery, Washington University, One Barnes Hospital Plaza, Suite 11300 West Pavilion, St. Louis, Missouri 63110, USA.

From PubMed study Gene-mediated osteogenic differentiation of stem cells by bone morphogenetic proteins-2 or -6.

J Orthop Res. 2006 Jun;24(6):1279-91. – Zachos TA, Shields KM, Bertone AL.

Me: I found these three studies which gave me another idea on how to possibly lengthen the long bones. The issue with this proposed method is that even in theory, there is only a small chance that it can increase the long bone’s length and only by a little, if the gene therapy is the only thing used. If we can combine the gene therapy method involved with a form of mechanical loading or invasive surgical distraction, the chances of achieving long bone lengthening will be increased dramatically.

Description & Analysis: What we see here is that genetically engineered pluripotent human mesenchymal stem cells can be grown and implanted with certain types of vectors which mutates them completely and causes them to overexpress on certain types of genes. In the first study, the already rhBMP-2 cells are injected even further with a new type of compound I am not familiar with, a lacZ gene, that encodes for beta-galactosidase. The researchers conclude very clearly that “the ability of GEPMC to engraft, differentiate, and stimulate bone growth“. If the GEPMCs can be mutated in vitro to have certain types of characteristics, why can we not just use gene therapy to cause the cells to overexpress chondrogenetic growth factors? In the 2nd study, we see that the vector used was to encapsulate BMP-2. After that the researchers injected doxycycline to control the growth of  bone. This means that after we get the first injection of MSCs in, that does not mean that we can do nothing to control the chondrogenesis anymore. Using doxycycline, we can cause the cells to probably be even more effective in the gene expression. The 3rd study state conclusively that “they can be genetically engineered to express desired therapeutic proteins inducing specific differentiation pathways.”

Actual Method: The method is still invasive because it has to penetrate tissue and bone. Let’s assume the idea of LSJL is valid. That means that as long as we can get cell differentiation towards chondrogenesis, proliferation, and hypertrophy, then the bones can lengthen. I then proposed that we first use gene therapy to mutate at least 2 batches of a certain type of MSCs. We first extract the MSCs of the patient from their long bone to get the re-implant to be immunologically compatible. We then add one type of vector for each of the two groups. For the first batch of mutated MSCs, we use vectors to get to cause them to focus on only expressing chondrogenesis genes. Once we accomplish that, we inject this first group of cells into the epiphysis ends of the long bones through drilling. After the cells are added, we can wait for 1-4 weeks. After we are sure the differentiation is complete, we want to expand the number of chondrocytes inside. That is when we get the 2nd group of MSCS to focus on overexpressing the genes that produce the growth factors that lead to chondrocyte proliferation. This in theory should not only cause the already chondrocytes implanted to multiply, it should also get the original MCSs in the epiphysis to differentiate as wel and join with the implanted groupl. Sure, we could make the argument that only the 2nd group of implants are needed, but from what I have learned about crystal growing ,it is always  better idea to start with a seed, and have it grow bigger and bigger but getting the surrounding medium and raw material to transform into it and then layer on top of it. Once this is done, there will be a substantial amount of chondrocytes in the human epiphysis. Since LSJL theory says induced chondrogenesis proliferation will lead to bone lengthening, then this proposed method should lengthen the bone. We then can use compounds like doxycyline, to control the rate of bone growth. To help the bone growth further along by causing chondrocyte aggregation, we can then apply ESW therapy in that area to help the Chondrocytes already there to lead to hypertrophy and expand.

From PubMed study 1 link HERE…

J Gene Med. 1999 Mar-Apr;1(2):121-33.

Engineered pluripotent mesenchymal cells integrate and differentiate in regenerating bone: a novel cell-mediated gene therapy.

This post is to focus on the cellular and orthopedic mechanics. One of the best things about this post is to see which articles and studies it has been using as a reference and some of the studies it does cite are extremely insightful on what may be the next approach to get the bone’s to respond correctly.

Analysis & Interpretation

It seems the researchers already know that long term stimulation through some type of mechanical or biological means of the bone marrow derived stem cells (BMSCs) causes them to differentiate into bone cells. Now they want to see from this study where just a small short stimuli would also work in stimulating BMSC differentiation into osteocytes.

I have posted the entire study below but I would only be going over the abstract and conclusion since those parts are the most critical. The amount of loading that was done was 24 hours of cyclic loading at a frequnecy of 0.05 HZ and force/area load of 4 kilapascals. The BMSCs where placed in a type of scaffold/solid porous medium and then solid was hit by the mechanical dynamic load. The BMSCs started to turn on/express the right type of genes that cause them to differentiate/change into bone cells.

There are the growth factors that increased

• Osteopontin
• Integrin – Beta1
• Transforming growth factor – Beta receptor 1
• SMAD
• Platelet derived Growth Factor -alpha
• annexin -V

The researchers conclude that the main reason these growth factors increased is from the cyclical loading on the bone. As stated, “it may be postulated that short-term mechanical stimulation results in an improved osseous integration of tissue engineered grafts in bone defect healing”

Implications For Height Increase

It has shown quite conclusively that for bone formation, the cyclical dynamic loading that is done to the stem cell holding fibrillin matrix material causes the progenitor cells to be more osteogenic in nature. The real question now is to see whether the same type of compressive loading will cause the same for cartilage formation. and if it does lead to chondrogenesis, in what ratio to bone formation. We have found now so many ways that are non-invasive to stimulate bone growth but the bone growth is for denstiy, but not volume increases longitudinally.

From PubMed study link Osteogenic Predifferentiation of Human Bone Marrow-Derived Stem Cells by Short-Term Mechanical Stimulation

[Note: This post is a resource post which only has information to help me build a atronger understanding on the effects of physical activity and loading on the epiphyseal growth plates.]

If you really wish to join me and the other height increase researchers, this page would be a very post to read and fully understand. The main thing to take away is that too much loading can disrupt the endochondral ossification process and cause fracture in the cartilage for young kids. Too little physical activity seems to decrease the bone density and cause the ability of the estrogen released during the initial part of puberty to not cause as great of chondrocyte hypertrophy (thus height increase) than if the young child does get some degree of physical activity.

The information below was taken from the website for the Journal of Clinical Medicine Research, Vol. 3, No. 1, Feb 2011. The source link is from HERE…

[Note: There was a References section below the post below which I decided not to copy and paste below]

Journal of Clinical Medicine Research, Vol. 3, No. 1, Feb 2011

The Effects of Physical Activity on the Epiphyseal Growth Plates: A Review of the Literature on Normal Physiology and Clinical Implications

Timothy A. Mirtz^a, c, Judy P. Chandler^b, Christina M. Eyers^b

^aDivision of Health Physical Education and Recreation, University of South Dakota, Vermillion, South Dakota, USA
^bDepartment of Physical Education and Sport, Central Michigan University, Mt. Pleasant, Michigan, USA
^cCorresponding author: 221B Dakota Dome, 414 East Clark Street, University of South Dakota, Vermillion, South Dakota 57069, USA. Email: timothy.mirtz@usd.edu

Manuscript accepted for publication November 23, 2010
Short title: Physical Activity on Epiphyseal Growth Plates
doi:10.4021/jocmr477w

Abstract

Background: Children need physical activity and generally do this through the aspect of play. Active play in the form of organized sports can appear to be a concern for parents. Clinicians should have a general physiological background on the effects of exercise on developing epiphyseal growth plates of bone. The purpose of this review is to present an overview of the effects of physical activity on the developing epiphyseal growth plates of children.

Methods: A National Library of Medicine (Pubmed) search was initiated using the keywords and combinations of keywords growth plate, epiphyseal plate, child, exercise, and physical activity.

Discussion: Bone is a dynamic tissue with a balance of osteoblast and osteoclast formation. The normal functioning of the epiphyseal growth plate is an important clinical aspect. Much of the physiology of the epiphyseal growth plate in response to exercise includes the important mechanical component. Growth hormone, insulin-like growth factor I, glucocorticoid, thyroid hormone, estrogen, androgen, vitamin D, and leptin are seen as key physiological factors. While there is a need for children to participate in physical activity, clinical consideration needs to be given to how the epiphyseal growth plate functions.

Conclusions: Mechanical loading of the bone is important for epiphyseal plate physiology. Exercise has a healthy function on the normal growth of this important biomechanical feature. Clinically, over-exertion in the form of increased load bearing on the epiphyseal growth plate creates an ideal injury. There is a paucity of research on inactivity on the epiphyseal growth plate resulting in stress deprivation. Further research should take into consideration what lack of exercise and lessened mechanical load bearing has on the function of the epiphyseal growth plate.

Keywords: Child; Physical activity; Epiphyseal growth plates; Bone; Exercise; Mechanical loading

Introduction

Bone has been described as a dynamic and highly interactive complex of many cell and tissue types [1]. The epiphyseal growth plate is made of several key aspects including cartilaginous,bony, and fibrous components, which act together to achieve longitudinal bone growth [2]. The epiphyseal growth plate is a final target organ for longitudinal growth and results from the cellular process of chondrocyte proliferation and differentiation [3]. Jaramillo and Hoffer [4] described the cartilaginous structures at the ends of growing bones as constituting the “growth mechanism. Frost [5] noted that knowledge of epiphyseal growth plate physiology has application for several areas. The first area noted being that such knowledge aids in distinguishing mechanically competent bone from incompetent bone [5]. The second area is that this knowledge enables a person to increase and maintain bone strength during growth [5]. Finally,knowledge that bone strength, taken in its entirety, and bone health are to be understood as completely different from each other with regards to physiological responses [5]. Of particular importance to clinical practice is the knowledge that two major contributions to the development of articular cartilage are growth factors and mechanical loading [6].

The purpose of this review is to present an overview of the effects of physical activity on the developing epiphyseal growth plates of children. A discussion of the physiological basis of epiphyseal growth plates will be included. Practitioners with a familiarity of the dynamic changes that can occur with the epiphyseal plate in normal children can ultimately lead to recognition of pathologic states [7, 8].

A Review of the Normal Physiology of the Epiphyseal Growth Plate

As noted previously, skeletal growth at the epiphyseal plate is an active and dynamic process [8]. The epiphyseal growth plate, being a highly specialized layer of cartilage where chondrocytes proliferate and differentiate, brings forth longitudinal bone growth [9]. The epiphyseal growth plate can be divided into three main chondrocyte subpopulations: the resting,proliferating and hypertrophic chondrocytes [10].

Longitudinal bone growth is primarily achieved through the action of chondrocytes in the proliferative and hypertrophic zones of the growth plate [11]. Longitudinal bone growth occurs in the epiphyseal growth plate through a process called endochondral bone formation and ossification [12-14]. Endochondral bone formation is a process where resting zone chondrocytes are recruited to start active proliferation and then undergo differentiation, followed by mineralization [12]. Production of metaphyseal cancellous bone and growth in length are both linked to endochondral ossification with growth plate cartilage cell proliferation as the driving force [15].

Within the epiphyseal growth plate, chondrocyte proliferation, hypertrophy, and cartilage matrix secretion result in chondrogenesis [14]. Endochondral bone development leading at the epiphyseal growth plate contributes to longitudinal bone growth through a process through which undifferentiated mesenchymal cells differentiate into chondrocytes, which then undergo well-ordered and controlled phases of proliferation, hypertrophic differentiation, death, blood vessel invasion, and finally replacement of cartilage with bone [16]. The newly formed cartilage is invaded by blood vessels and bone cells that remodel the newly formed cartilage into bone tissue [14].

The regulation of linear bone growth in children and adolescents comprises a complex interaction of hormones and growth factors [17]. This process of longitudinal bone growth is governed by an intricate network of endocrine signals, including growth hormone, insulin-like growth factor I, glucocorticoid, thyroid hormone, estrogen, androgen, vitamin D, and leptin [14]. Many of these signals act locally on growth plate chondrocytes to regulate epiphyseal growth plate function [14]. The regulation of longitudinal growth at the epiphyseal growth plate occurs generally through the intimate interaction of circulating systemic hormones and locally produced peptide growth factors which has the net result of triggering changes in gene expression bygrowth plate chondrocytes [13].

In particular, for the majority of skeletal elements to develop and grow, the process of endochondral ossification requires a constantly moving interface between cartilage, invading blood vessels, and bone [1]. An adequate supply of calcium is critical for normal epiphyseal growth plate development and that changes in extracellular calcium modulate the function of chondrocytes with high calcium [Ca2+] leading to cell differentiation [18]. The active metabolite of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D), is classically appreciated to exert its calcemic and other actions via interaction with the vitamin D receptor thus modulating gene transcription [19]. However, with respect to parathyroid gland function and development of the cartilaginous epiphyseal growth plate, calcium and 1,25(OH)2D act cooperatively and 1,25(OH)2D will act independently of the vitamin D receptor [19].

The balance between proliferation and differentiation in bone is considered to be a crucial step.  It is a crucial regulatory step controlled by various growth hormones acting in the endocrine pathways [12]. Growth hormone (GH) and insulin-like growth factor-I have major effects on the chondrocytes of the growth plate and act upon all bone cells [20]. Growth hormone (GH), mostly seen in action during the growth spurt in early adolescence, is considered to be the key hormone regulator of linear growth during childhood [17].

Research with laboratory animals has provided most of the current information regarding estrogens influence on the growth process of long bones, on the maintenance of cancellous bone mass, and on the architectural and cellular changes in bone [21]. Estrogen action is indispensable for normal pubertal growth and growth plate fusion. Both estrogen receptors (ER), ER-alpha and ER-beta, are expressed in the growth plate in boys and girls throughout pubertal development [12]. The rise in estrogen levels at menarche in girls is associated with a large reduction in bone turnover markers and reflects the closure of the epiphyseal growth plates, the reduction in periosteal apposition and endosteal resorption within cortical bone [22].Jarvinen [23] noted that estrogen tends to pack mechanically-excess mineral into the female skeleton at puberty thus creating the paradigm that the most responsive period of female bone to mechanical loading occurs prior to menarche. This knowledge has consequence for the encouragement of physical activity especially for the female population.

As previously noted, longitudinal growth of the skeleton is a result of endochondral ossification that occurs at the epiphyseal growth plate. Through the sequential process of cell proliferation, extracellular matrix synthesis, cellular hypertrophy, matrix mineralization, vascular invasion, and eventually apoptosis, cartilage continually is being replaced by bone as length increases [15]. Parfitt [15] explained that genetic determination of bone mass is mediated by two classes of gene. The first class of gene (under the control of the sizostat) regulates growth of muscles and bones [15]. The second class of gene (under the control of the mechanostat) regulates the increase in bone density in response to load bearing [15]. With skeletal maturity, there is a decreased growth rate and is mainly associated with structural changes in the physis, including a gradual decline in growth plate width due to the reduced height of the proliferative and hypertrophic zones [13].

Mechanical Influence on the Physiology of the Epiphyseal Growth Plate

Comprehension of the biomechanical aspects of bone allows one to conceptualize the physiological processes associated with exercise and physical activity on the epiphyseal growth plate.The mechanical influence on bone directly applies to the normal physiological functioning of bone. Longitudinal growth is controlled by local mechanical factors in the form of a feedback mechanism which exists to ensure that bone growth proceeds in the direction of the predominant mechanical forces [11]. The individual epiphyses undergoes a characteristic series ofevents; central calcification, absorption of cartilage and endochondral ossification, the further course of which is definitely determined by the degree of local distortion [24]. Production of diaphyseal cortical bone and growth in width are both linked to periosteal apposition driven by the process of osteoblast precursor proliferation [15]. During adolescence the trabeculae and cortices become thicker by endosteal apposition which increases bone density [15]. Intrinsically, biophysical forces placed upon the bone assist to develop the growing bone while extrinsic biophysical forces tend to resist and channel the expansion of bone into its functional forms such as the internal trabecular architecture and the external shape [25].

In addition to the vital function of growth factors, it is known that mechanical forces stimulate the synthesis of extracellular proteins in vitro and in vivo and can affect the tissue’s overallstructure [7]. According to the mechanostat theory, periosteal apposition is regulated by biomechanical requirements [11]. The stress acting on the cartilaginous epiphysis is comparable tothat in the adult with relative differences attributed to variations in the mechanical relationship and to the hormonal control of the body’s growth [24]. Frost [5] noted that later-discovered tissue-level mechanisms and functions (including biomechanical and muscle) are the true key players in bone physiology, and homeostasis ranks below the mechanical functions.

The Role of Exercise and Physical Activity on the Epiphyseal Growth Plate

Over the past decade, there has been a surge in the number of sports opportunities available to young athletes [26]. Beyond the positive physiological, psychological and social aspects that a sports activity brings to adolescents, there exists the potential risk of injuries and overuse of the locomotor system [27]. Nonetheless, pursuing sports as a leisure time activity has not been found to be harmful for children [28]. However, this must be coupled with proper instruction, appropriate amounts of physical activity as well as safety.

The effects of exercise on the molecular nature of secreted human growth hormone (GH) or its biological activity are not well understood [29]. Yet it is known that children have more elastic soft tissue and more potential for remodeling than adults [30]. Epiphyseal growth plates are often less resistant to deforming forces than ligaments or joint structures. A child’s skeletal system shows pronounced adaptive changes to intensive sports training [31, 32]. The growing skeleton is said to be more responsive than the mature skeleton to the osteotrophic effect of exercise [14]. The mechanstat, a process which is mediated by the detection of deviations from a target value for strain and is a conglomeration of cellular responses that tend torestore target values [15]. From this, the genetic influence on bone mass and density are largely mediated by body size, bone size, and muscle mass [15]. Most long bones end near the joint in a separate epiphysis which at first consists of cartilage and is later ossified [24]. This epiphysis becomes fused with the shaft of the bone and in most cases only at the end of puberty [24]. Thus the stage of growth and development of the child is suggestive of the amount and intensity of exercise that can be performed and tolerated. The recommendation that children, especially adolescents should not perform activities such as plyometrics or engage in heavy weight lifting and should concentrate mainly on such activities that encourages high repetitions at a very low weight [33].

Vascularization of the epiphyseal growth plate region represents a key mechanism for the coupling of two fundamental processes determining the rate of bone growth: chondrogenesis(cartilage production) and osteogenesis (bone formation) [34]. Within the epiphyseal cartilages of such anatomical entities such as the capitellum, trochlea, and medial and lateral epicondyles, vascularity is centripetal [35]. Due to the vascularity pattern it is difficult for avascular necrosis to develop after trauma within the epiphyses [35]. This type of vascularity would represent intraosseous vascularity whereas blood supply outside the epiphyseal plate would represent extraosseous vascularity. This is due in part that there is no true blood supply to the physis but rather the blood supply advances from the blood vessels of the epiphysis and metaphysis and from the perichondrial ring and vessels of the periosteum [36].

Influence of Over-activity on the Epiphyseal Growth Plate

A potential problem with physical activity and exercise on the epiphyseal plates is over-activity. Intuitively, it is the extent that over-physical activity may have on the growth plate resulting in injury. A better appreciation of how epiphyseal plate physiology works is seen in the response to trauma.  Approximately 25% of adolescents have at least one recreational injury which is mostly minor reflecting only soft tissue trauma and abrasions of the skin [37]. However, approximately 15% of children with fractures involve physeal injuries with 10% of these physeal injuries being sport-related [36]. The most prevalence of epiphyseal growth plate injuries is to children ages 10 to 16 years [38, 39]. If injury occurs to the epiphyseal growth plate the possibility that there may be a premature locking of the epiphyseal growth plate essentially halting bone lengthening [40].

Most injuries in childrens sports and activities are minor and self-limiting thus suggestive that children and youth sports are safe [32]. Unlike adults, many of the injuries may be treated closed due to the growth and remodeling potential of children [41]. Skeletally immature children who participate in extreme levels of sports participation can sustain repetitive trauma[42]. This repetitive trauma can cause the epiphyseal plate to widen. Laor et al [42] hypothesized that the  metaphyseal vascular supply is disrupted causing the normal process of endochondral bone formation due to long columns of hypertrophic cartilage cells from the physis extending into the metaphysis  As the risk of injuries sustained by young athletes can be significant, it is essential that training programs take into account physical and psychological immaturity, so that the growing athlete can adjust to their own body changes [31]. The period of early puberty is associated with an increased risk of fracture which may be related to the high rate of bone turnover [22]. A late menarche is a consistent risk factor for fracture in young females due to hormonal instability that may affect bone density [22]. Growth disturbance depends on extent of the injury and the amount of remaining growth potential [36].However, it can be hypothesized that a partially closed physis are weak links in children and that asymmetrically or partially closed physis may be vulnerable to trauma [43]. It has been suggested that overuse during puberty for females may result in long term development of low bone density and ongoing problems with bone health.

The genetic potential for bone accumulation can be frustrated by insufficient calcium intake, disruption of the calendar of puberty and inadequate physical activity [15]. While many of the molecular mechanisms that control cellular differentiation and growth during embryogenesis recur during fracture healing taking place in a post-natal environment that is unique and distinct from those which exist during embryogenesis [44]. Disruption of either the longitudinal intraosseous vasculature (vertical extraosseous blood supply) or the vascular arch in morethan two places may lead to selective avascular necrosis (extraosseous) of the epiphyseal cartilage [35].

The clinical pathophysiology of excessive activity on the epiphyseal growth plates resulting in injury is one of the more prominent methods of understanding the effects of exercise on growth plate physiology. The immature skeleton is different from the adult skeleton with unique vulnerability to acute and chronic injuries at the growth plate [7]. Epiphyseal injuries are usually due to shearing and avulsion forces as well as compressive forces usually due to either severe twisting or direct blows that can result in a disruption of the epiphyseal growth plate[31]. In young athletes, as the bone stiffness increases and resistance to impact diminishes, sudden overload may subject bone to either bow or buckle [31]. Swelling, hyperemia, and deformity in the physeal area are the classical signs of physeal injury with pain being potentially less intense [36]. Physeal injuries occur in 15% of children with fractures, and 10% of all physeal injuries are sport-related [36].

Gerstenfeld et al [44] summarized five key points of damaged epiphyseal plate healing that needs to be considered clinically. First, the anatomic structure of callus formation as it progresses during the healing phases should be considered [44]. Second, morphogenetic signals that facilitate the repair process should be known [44]. Third, and of importance for clinicians, is the role of the biomechanical aspect in controlling differentiation during cellular repair [44]. Fourth, the role of key groups of soluble factors i.e. pro-inflammatory cytokines and angiogenic factors during repair are important for establishing vascularity [44]. Finally, knowledge and appreciation for the relationship between the genetic components that control bone mass and remodeling is warranted [44]. These five key points should be acknowledged clinically in an effort to monitor for proper healing post-injury to the epiphyseal plate. From these key points, one can gain an appreciation for not reducing sport activity but in the intensity of the sport activity during the high intense growth phases. For example, it is not uncommon to see adolescents training year-round in one specific sport without the ability to either rest during the off-season or enter another sport that may be different and/or less intense in the training. As well, prohibitions of negative training and in some cases, the prohibition of sports all together, are sometimes necessary to minimize the potential for injury [27].While sports are important for children, safety and prevention of needless injury should be considered.

Influence of Inactivity on the Epiphyseal Growth Plate

The second and arguably the least discussed aspect concerning the epiphyseal growth plate is the role inactivity may play on the growth plate. Given the current interest in and rising rates of child obesity such interest in the growth plate should be considered. One of the implicated culprits in the child obesity epidemic is the lack of physical activity. It is known that epiphyseal growth plate activity controls longitudinal bone growth and leads ultimately to adult height yet numerous disorders are characterized by retarded growth and reduced final height eitherhave their origin in altered chondrocyte physiology or display pathological growth plate changes secondary to other causes [45]. Physical activity may have a protective effect on the epiphyseal growth plate, however, very little research has been conducted on the role of inactivity on the epiphyseal growth plates. This is possibly due to the lack of clinically-related biomechanical problems that emanate from a lack of physical activity on the epiphyseal plates. In other words, there is a paucity of research in the form of case studies that has implicated a lack of physical activity as an etiology for epiphyseal growth plate injury. Despite this, one could hypothesize that physical inactivity would not serve as an effect mechanism of protection. Frost [5] observed this very phenomenon but noted that for an obese person the stronger muscles would put larger loads on bones to which bone physiology should respond by increasing bone strength even if non-mechanical factors are involved. Until further research is undertaken on the effects of inactivity on the growth plate, one can extrapolate such effects from the current literature on normal and excessive functioning that sedentarism may result in inadequate stimulation of the growth plate with a possible result of changed growth potential.

Nonetheless, the recent concern over the increasing incidence and prevalence of obesity as seen in children gives rise to concern for the normal growth process. As previously indicated,functioning growth hormone (GH) and insulin-like growth factor (IGF)-I are essential for normal growth [46]. However, obese children will typically grow at a normal rate despite the presence of low serum GH levels with leptin, insulin, and sex hormones working to locally activate the IGF system at the epiphyseal plate [46]. Serum leptin may play a biological role in regulating bone metabolism by increasing the proliferation and differentiation of osteoblasts in adults [47]. Phillip et al [46] found that an elevated level of leptin in obese children can affect the bone growth center and it may be that leptin also participates in growth without GH observed in obesity.

It may appear that, in some cases, genetic expression, through favorable conditions, can be maximally achieved throughout the entire period of growth [48]. In this instance, it is hypothesized that for harm to be placed on the growth plate or show delayed growth maturation at the epiphyseal growth plate for those children who are physically inactive, thecombination of genetic expression through unfavorable environmental and socioeconomic conditions may be the culprit in abnormal epiphyseal growth plate physiology. In other words,only through unfavorable conditions such as extreme poverty, lack of nutrition, and other entities will the genetic expression manifest itself for those children who are physically inactive.While it is known that load-bearing tissue, such as articular cartilage, will atrophy in the absence of mechanical forces [6], future investigation into the effects of a lack of load bearingthrough a lack of physical activity on the epiphyseal plate is warranted.

Conclusion

The epiphyseal growth plate is a dynamic entity. Growth is dependent not only on intrinsic factors such as hormones and other regulatory factors but on extrinsic factors. These extrinsic factors are based entirely on the biomechanical model. Exercise, a positive aspect for the epiphyseal growth plate needs to be moderated through carefully crafted activities especially during pubertal growth spurts. Obesity, a major problem among todays youth, can be attributed in part to a lack of exercise. Once activity is undertaken the potential for epiphyseal growth plate disturbances from too much activity may be a predisposing factor to growth plate dynamics. The effects of exercise on the epiphyseal growth plate needs further research to comprehend the entirety of this dynamic anatomical and physiological entity. Research in the area of the epiphyseal growth plate in some children who are sedentary needs to be addressed.

Authors Contributions

All authors contributed equally to the conceptualization, writing, and approval of the paper.

Competing Interests

The authors declare no competing interests.