Growth Plate transplants not so far fetched

Note that I believe that hydrostatic pressure induced chondrogenic differentiation of stem cells is currently the best way to grow taller.  That doesn't mean the science of stem cells in growth plate repair can't help us in our quest for height. 

Repair of injured articular and growth plate cartilage using mesenchymal stem cells and chondrogenic gene therapy. 

"The potentials of mesenchymal stem cells (MSCs) in cartilage repair include (a) identifying readily available sources of and devising appropriate techniques for isolation and culture expansion of MSCs that have good chondrogenic differentiation capability, (b) discovering appropriate growth factors (such as TGF-beta, IGF-I, BMPs, and FGF-2) that promote MSC chondrogenic differentiation, (c) identifying or engineering biological or artificial matrix scaffolds as carriers for MSCs and growth factors for their transplantation and defect filling[if they identify some carriers that are already in the body we could take advantage of that to gain height]. Gene therapy with chondrogenic growth factors or inflammatory inhibitors (either individually or in combination), either directly to the cartilage tissue or mediated through transducing and transplanting cultured chondrocytes, MSCs or other mesenchymal cells [can be used to regenerate cartilage]. [What is] the optimal combination of MSC sources, growth factor cocktails, and supporting carrier matrixes? As more insights are acquired into the critical factors regulating MSC migration, proliferation and chondrogenic differentiation both ex vivo and in vivo, it will be possible clinically to orchestrate desirable repair of injured articular and growth plate cartilage, either by transplanting ex vivo expanded MSCs or MSCs with genetic modifications, or by mobilising endogenous MSCs from adjacent source tissues such as synovium, bone marrow, or trabecular bone." 

What we're doing with LSJL is mobilizing endogenous MSCs from the bone marrow. 

Let's look at the three criteria for successful growth plate cartilage repair(and by extension successful formation of new growth plate cartilage): 

1) MSCs capable of chondrogenic capacity.  This should be true of bone marrow MSCs as they are capable of mesenchymal chondrosarcoma(which involves chondrogenic differentiation). Telomere Length and Methylation Status play a role also. 

2) The second factor is a way of encouraging chondrogenic differentiation.  This is hydrostatic pressure.  Which we induce by laterally compressing the bone to increase the fluid pressure within the bone marrow.  There are alternatives to this such as LIPUS plus TGF-Beta.  

3)The scaffolds for transplantation or direct filling.  Well, the MSCs are already in the body so there's no need for transplantation or filling.  They are already in a position to increase height. 

In vitro stage-specific chondrogenesis of mesenchymal stem cells committed to chondrocytes.

"A coculture preconditioning system was used to improve the chondrogenic potential of human MSCs using a human MSC line, Kp-hMSC, in commitment cocultures with a human chondrocyte line, hPi (labeled with green fluorescent protein [GFP])[MSCs were co-cultured with chondrocytes]. Committed MSCs were seeded into a collagen scaffold[bone is technically already a collagen scaffold Type II collagen] and analyzed for their neocartilage-forming ability.
Coculture of hPi-GFP chondrocytes with Kp-hMSCs induced chondrogenesis, as indicated by the increased expression of chondrogenic genes and accumulation of chondrogenic matrix, but with no effect on osteogenic markers. The chondrogenic process of committed MSCs was initiated with highly activated chondrogenic adhesion molecules[studying these chondrogenic adhesion molecules could be a way to help us gain height] and stimulated cartilage developmental growth factors, including members of the transforming growth factor beta superfamily and their downstream regulators, the Smads, as well as endothelial growth factor, fibroblast growth factor, insulin-like growth factor, and vascular endothelial growth factor. Committed Kp-hMSCs acquired neocartilage-forming potential within the collagen scaffold.
Human MSCs committed to the chondroprogenitor stage of chondrocytic differentiation undergo detailed chondrogenic changes."

Now, LSJL can't co-culture MSCs with growth plate chondrocytes as there are no growth plate chondrocytes left after fusion.  However, LSJL can induce chondrogenesis by other means.  This does mean that maybe pre-fusion that something like LIPUS can increase height on it's own as LIPUS causes shear strain in the bone disrupting the actin cytoskeleton and could allow for adhesion of MSCs to chondrocytes. 

Application of autologous bone marrow derived mesenchymal stem cells to an ovine model of growth plate cartilage injury. 

"Injury to growth plate cartilage in children can lead to bone bridge formation and result in bone growth deformities. Mesenchymal stem/stromal cells (MSC) offer a promising therapeutic option for regeneration of damaged cartilage, due to their self renewing and multi-lineage differentiation attributes. Our laboratory has recently characterised MSCs derived from ovine bone marrow, and demonstrated these cells form cartilage-like tissue when transplanted within the gelatin sponge, Gelfoam[Will have to explore height increase applications of Gelfoam], in vivo. In the current study, autologous bone marrow MSC were seeded into Gelfoam scaffold containing TGF-beta1, and transplanted into a surgically created defect of the proximal ovine tibial growth plate. Examination of implants at 5 week post-operatively revealed transplanted autologous MSC failed to form new cartilage structure at the defect site, but contributed to an increase in formation of a dense fibrous tissue. Importantly, the extent of osteogenesis was diminished, and bone bridge formation was not accelerated due to transplantation of MSCs or the gelatin scaffold." 

So the transplant failed to induce chondrogenesis and instead underwent fibrogenesis.  Note that they didn't use shear strain at all and from the previous research shear strain plus TGF-Beta is needed for chondrogenesis.  Also the gelatin scaffold may have been insufficient. 

"Analysis of growth plate defects treated with Gelfoam scaffold and autologous MSC revealed a tissue composition consisting of dense fibrous (38.2 ± 6.1%), fibrous (29.6 ± 10.0%), fat (24.2 ± 6.6%) and bone (7.9 ± 1.8%)."

Also they inserted the growth plate scaffold directly into the growth plate defect that may be a part of the problem and they would've had better luck with a scaffold in a bone fracture.

But at least the technology is there.  They just need better ways to induce chondrogenesis.  Of course, you could just do LSJL. 

Here's a growth plate transplant experiment in action:


Assessment of epiphyseal plate allograft viability and function after ex vivo storage in university of wisconsin solution.

"Compromised epiphyseal plate function can result in limb deformities. Microvascular transplantation of an epiphyseal plate allograft is a potentially effective approach to reestablish longitudinal limb growth. The goal of this study was to determine a time frame for which proximal tibial epiphyseal plate allografts could be stored in University of Wisconsin Preservation Solution (UWPS) and remain functional in vivo after microvascular transplantation.

Proximal tibial epiphyseal plate allografts from skeletally immature female New Zealand White rabbits (10 to 12 wk of age) were used. Allografts (isolated on the popliteal arteriovenous pedicle) were stored ex vivo in cold UWPS for periods of up to 21 days. Chondrocyte viability, phenotype, and extracellular matrix composition of growth plate cartilage was assessed. Microvascular transplantations of nonstored or prestored (3 d) allografts were performed and analysis of bromodeoxyuridine and calcein incorporation was done to determine chondrocyte proliferation and new bone growth, respectively.

In vitro analysis showed that, compared with control tissue, epiphyseal plate chondrocyte viability, organization, and collagen extracellular matrix was preserved up to 4 days in cold UWPS. Microvascular transplantation of nonstored epiphyseal plate allografts was successful[scientists can transfer growth plates that have not been stored]. Despite care being taken to ensure vascular patency during the microvascular procedure, transplantation of prestored allografts failed due to absent flow in the larger vessels and in the allograft based upon the visualization of organized thrombus within the vascular pedicle, and absent flow within the composite graft itself. However, growth plate viability and function was detected in a peripheral region of a single allograft where partial blood flow had been maintained during the transplantation period.

Ex vivo storage in cold UWPS for 3 days maintains growth plate chondrocyte viability and function in vivo. However, future studies must be directed toward investigating the direct effect of ex vivo storage on the integrity and function of the vascular pedicles."

So growth plate transplantation is possible along as the growth plate is not in storage for more than 3 days.  Although I don't see why you wouldn't just differentiate a new growth plate in the bone marrow.

Here's a study related to formation of new growth plates so they can be available for transplant:

Fetal Mesenchymal Stromal Cells Differentiating towards Chondrocytes Acquire a Gene Expression Profile Resembling Human Growth Plate Cartilage.

"We used human fetal bone marrow-derived mesenchymal stromal cells (hfMSCs) differentiating towards chondrocytes as an alternative model for the human growth plate (GP). [Are] chondrocytes derived from hfMSCs are a suitable model for studying the development and maturation of the GP? hfMSCs efficiently formed hyaline cartilage in a pellet culture in the presence of TGFβ3 and BMP6.  A set of 232 genes was found to correlate with in vitro cartilage formation. Several identified genes are known to be involved in cartilage formation and validate the robustness of the differentiating hfMSC model. KEGG pathway analysis using the 232 genes revealed 9 significant signaling pathways correlated with cartilage formation. We compared the gene expression profile of differentiating hfMSCs with previously established expression profiles of epiphyseal GP cartilage. As differentiation towards chondrocytes proceeds, hfMSCs gradually obtain a gene expression profile resembling epiphyseal GP cartilage. We visualized the differences in gene expression profiles as protein interaction clusters and identified many protein clusters that are activated during the early chondrogenic differentiation of hfMSCs showing the potential of this system to study GP development."

"Pellet cultures were used to induce chondrogenic differentiation of hfMSCs"

"The mean diameter of the pellets increased with time, as well as the amount of glycosaminoglycans, a major constituent of the cartilaginous extracellular matrix. Immunofluorescent staining for collagen type II demonstrated the presence of chondrocytes after 1 week of pellet culture. The expression of collagen type II increased over time. Hypertrophic chondrocytes were first detected after 3 weeks, as evidenced by immunohistochemical staining for collagen type X. These collagen type X positive cells were located in a discrete ring-like zone surrounded by collagen type II positive chondrocytes. In all stages of differentiation, the chondrogenic core of the pellets was surrounded by a thin layer of two to three undifferentiated cells"

"Global gene expression microarray analysis showed that the Wnt antagonist DKK1 and FRZB and the BMP antagonist GREM1 are highly expressed in articular cartilage as compared to growth plate cartilage."

"PANX3, EPYC{up 6 fold in LSJL}, WNT11 and LEF1 are highly expressed in growth plate cartilage as compared to articular cartilage"

KEGG Pathways expressed in growth plates:

Focal Adhesion

Cytokine and Cytokine Receptor Interaction

Wnt and IHH signaling

Complement and coagulation

TGFBeta Signaling

Cell Communication and Extracellular Matrix Interaction

B-cell receptor signaling

Click on the image to see it enlarged.

"Analysis of protein interactions of all genes that were ≥3.29-fold changed after 5 weeks of chondrogenic differentiation as compared to undifferentiated hfMSC"

Genes changed that were not in highlighted clusters in diagram also altered in LSJL:

CAPN6{up}

LRRC1{down}

CADM1{down}

ITGBL1{up}

ARL6ip1{down}

Acta2{up}

Angptl1{up}

Tmem100{up}

Scn3a{up}

Slc38a4{up}

Dpt{up}

Cluster A(all genes listed genes that were altered by LSJL are noted as these genes may not have been altered by LSJL immediately but may have been altered by LSJL at a later time point or at under 2 fold.  Bolded means that the genes are centrally located in the cluster thus if they are altered by LSJL it's more likely that the other genes are too):

ADAMTS1{up}

OMD

GDF5

Sp7

Acan{up}

Epyc{up}

Col10a1{up}

Adamts5

Col9a2

Ptprz1

Cntnap1

Ctsb

Fap

Comp

Col9a3{up}

KIAA1199

Slc24a2

CHAD

PTH1R

Spp1

Col2a1{up}

Chi3l1

Fmod

Col11a1{up}

29.2%

Cluster B:

S100P

TGFBR3

HSPA8

UQCRFS1

CAV1

CRLF1{up}

LRP4

Basp1{down}

Grem1

Has2

FST

Bambi

Wif1

15.4%

Cluster C:

ID3

Hey1

Slc14a1

F13a1

Wnt11

Vcam1{down}

Nqo1

Akr1c3

Serpina3

MMP1

Plau

Fos{up}

Jun{up}

Sox8

21.4%

Cluster D:

Ccnb1

Prc1

Ndc80

Pbk

Ube2c

Plk2

Ccnc

Orc6l

Dlgap5

Melk{down}

10%

Cluster E:

Pcolce2

Ogn{down}

Smoc2{up}

66%

LSJL likely alters expression of clusters A, C, and E.

Now here's human fetal MSCs gene expression versus growth plate gene expression:

"Analysis of protein interactions of genes that are differentially expressed in undifferentiated hfMSC (week 0) compared to average expression profiles of growth plate cartilage of 3 prepubertal donors "

No Cluster Genes that were altered in growth plate cartilage and LSJL:

Gldn{up}

Fxyd6{up}

BSP{up}

Tagln{up}

Postn{up}

Acta2{up}

Col16a1{up}

Vgll3{up}

Scn3a{up}

Fzd2{up}

Capn6{up}

Arl4c{down}

Tardbp{down}

Ankrd29{up}

Slc5a1{up}

Spp1{up}

Cluster A:

Acan{up}

Omd

Matn2{up}

Adamts1{up}

Frzb

MMP9

Ptprz1

Col9a2

Col10a1{up}

Matn3{up}

Epyc{up}

Csgalnact1

Cntnap1

Phlda2

Col2a1{up}

Sox9{up}

Comp

Matn1

Col11a1{up}

Col11a2

Fmod

MMP13

Ctsk

Chi3l1

Lect1

Itga10

Pth1r

FGFR3

CHAD

Prelp

Gprasp1{down}

Wisp3

Col9a1{up}

33.3%

Cluster B:

Grem1

Rgmb

Sost

Gasp1

Has2

Col15a1{up}

glipr1

bambi

fat3

daam1

Wif1

Tmemff2

C4orf49

7.7%

Cluster C:

MMP1

IGFBP3

GBP1

Fap

Tnfaip8{down}

Fos{up}

Fosb{up}

Serpina3

37.5%

Cluster D:

Clu

Serpine1{up}

Gas6

F13a1

Lpl

Lif

Loxl1

TNIK

Vegfc

Serpina1

PF4

Neto2

CH25H{up}

15.4%

Cluster E:

S100B

Myo5c

Prss23

Man1a1

Boc

Ptx3

Serpina5

CTCFL

Clgn

Rbp4

Ifit1

Cdh13{up}

Adipoq

Gpnmb

Sox8

Cxcl14

Smoc2{up}

Ogn{down}

Pcolce2

Hbb

Hbd

Hba2

F3

TF

IGFBP4

MYO5C

11.5%

Cluster F:

BDNF

Eno4

Uchl1

Scg2

Scrg1

Cnih3

Spry2

Stmn2

0%

LSJL likely alters clusters A and C.

"Growth and differentiation factor 5 (GDF5), previously reported as stimulator of chondrocyte proliferation, was highly expressed at the earliest time point observed and down regulated thereafter."

"Cartilage matrix analysis is often limited to examining collagen type 2 and aggrecan expression. These markers are characteristic for hyaline cartilage and cannot distinguish growth plate cartilage from articular cartilage. Indeed, Huang et al. performed global microarray analysis of adult bovine MSCs at time 0 and after 28 days of differentiation in agarose constructs and compared the gene expression profile to that of chondrocytes isolated from articular cartilage. They showed that chondrogenically differentiating MSC do not form articular cartilage at 28 days in the presence of TGFβ3"

Engineering osteochondral constructs through spatial regulation of endochondral ossification.

"Chondrogenically primed bone marrow derived mesenchymal stem cells (MSCs) have been shown to become hypertrophic and undergo endochondral ossification when implanted in vivo. Modulating this endochondral phenotype may be an attractive approach to engineering the osseous phase of an osteochondral implant. [We engineered] an osteochondral tissue by promoting endochondral ossification in one layer of a bi-layered construct and stable cartilage in the other. The top-half of bi-layered agarose hydrogels were seeded with culture expanded chondrocytes (termed chondral layer) and the bottom half of the bi-layered agarose hydrogels with MSCs (termed osseous layer). Constructs were cultured in a chondrogenic medium for 21 days and thereafter were either maintained in a chondrogenic medium, transferred to a hypertrophic medium, or implanted subcutaneously into nude mice. This structured chondrogenic bi-layered co-culture was found to enhance chondrogenesis in the chondral layer, appearing to help re-establish the chondrogenic phenotype that is lost in chondrocytes during monolayer expansion. The bi-layered co-culture appeared to suppress hypertrophy and mineralisation in the osseous layer. The addition of hypertrophic factors to the media was found to induce mineralisation of the osseous layer in vitro. A similar result was observed in vivo where endochondral ossification was restricted to the osseous layer of the construct leading to the development of an osteochondral tissue."

Fully differentiated chondrocytes do not undergo endochondral ossification upon implantation whereas MSCs derived into chondrocytes do.

"A structured co-culture of chondrocytes and MSCs significantly enhanced collagen synthesis in the top chondral layer of bi-layered engineered constructs compared to single layer constructs that only contained chondrocytes (133.32 ± 21.8 vs. 72.45 ± 18.63 ng/ng).  MSCs in single layer constructs accumulated significantly more collagen compared to MSCs in the bottom osseous layer of bi-layered constructs (154.65 ± 14.53 vs. 83.57 ± 21.38 ng/ng)."

"No evidence of mineralisation was observed in bi-layered constructs maintained in a hypertrophic medium without additional β-glycerophosphate supplementation (HM-). When β-glycerophosphate was added to the hypertophic medium (HM+), mineralisation of the osseous layer was observed"

"Both hypertrophic media formulations resulted in apparent elongation of the interface between the osseous and chondral layer of bi-layered constructs. sGAG accumulation in the chondral layer of the engineered tissue was significantly reduced for constructs maintained in HM+ compared to CM"

"Mineralisation of the osseous layer correlated with significant cell death as evidenced by a reduction in the DNA content in this layer of bi-layered constructs when cultured in a hypertrophic medium with additional β-glycerophosphate supplementation (HM+)"

"Mineral volume, was significantly greater for single layer MSC constructs compared to bi-layered constructs (6.09± 0.59 vs. 1.36 ± 0.42 mm3; n=3)"

"[There was] reduced type X collagen accumulation in the osseous layer of bi-layered constructs while type II collagen accumulation increased in the chondral layer."

"bi-layered coculture suppresses hypertrophy of MSCs and enhances chondrogenesis of chondrocytes"

"Single layer chondrocyte seeded constructs stained weakly for collagen type II, indicating that a certain degree of de-differentiation had occurred prior to hydrogel encapsulation."

"chondrogenically primed MSCs release growth factors and cytokines such as TGF-β3, BMP-2, IGF-1 and FGF-2"

"In hypertrophic media formulations, both with and without β-glycerophosphate supplementation, elongation of the interface between the two cell types was observed, suggesting perhaps that aspects of long bone growth are being mimicked in this culture system."