Monday, April 12, 2010

Red Bone Marrow Stem Cells, Lateral Synovial Joint Loading, and Growth Plates

Lateral Synovial Joint Loading increases interstitial fluid flow by lateral loading of the epiphysis of long bones.  Red bone marrow is present in the epiphysis of long bones.  Lateral Synovial Joint Loading provides a distraction force on the epiphyiseal plate of long bones.  Lateral Synovial Joint Loading transports red bone marrow into the growth plates.  Red bone marrow contains stems cells which can result in renewed growth plate activation.  Red Bone Marrow goes away as you age but it's still easier to inject new red bone marrow than to perform distraction osteogenesis(limb lengthing).

Let's study the effects of these red bone marrow stem cells and see if the transportation of them into the distracted(opened) growth plate can cause renewed growth.

Hypertrophy is induced during the in vitro chondrogenic differentiation of human mesenchymal stem cells[Red Bone Marrow stem cells] by bone morphogenetic protein-2 and bone morphogenetic protein-4 gene transfer.

"NTRODUCTION: The present study compares bone morphogenetic protein (BMP)-4 and BMP-2 gene transfer as agents of chondrogenesis and hypertrophy in human primary mesenchymal stem cells (MSCs) maintained as pellet cultures. METHODS: Adenoviral vectors carrying cDNA encoding human BMP-4 (Ad.BMP-4) were constructed by cre-lox combination and compared to previously generated adenoviral vectors for BMP-2 (Ad.BMP-2), green fluorescent protein (Ad.GFP), or firefly luciferase (Ad.Luc). Cultures of human bone-marrow derived MSCs were infected with 5 x 10(2) viral particles/cell of Ad.BMP-2, or Ad.BMP-4, seeded into aggregates and cultured for three weeks in a defined, serum-free medium. Untransduced cells or cultures transduced with marker genes served as controls. Expression of BMP-2 and BMP-4 was determined by ELISA, and aggregates were analyzed histologically, immunohistochemically, biochemically and by RT-PCR for chondrogenesis and hypertrophy . RESULTS: Levels of BMP-2 and BMP-4 in the media were initially 30 to 60 ng/mL and declined thereafter. BMP-4 and BMP-2 genes were equipotent inducers of chondrogenesis in primary MSCs as judged by lacuna formation, strong staining for proteoglycans and collagen type II, increased levels of GAG synthesis, and expression of mRNAs associated with the chondrocyte phenotype. However, BMP-4 modified aggregates showed a lower tendency to progress towards hypertrophy, as judged by expression of alkaline phosphatase, annexin 5, immunohistochemical staining for type X collagen protein, and lacunar size. CONCLUSIONS: BMP-2 and BMP-4 were equally effective in provoking chondrogenesis by primary human MSCs in pellet culture. However, chondrogenesis triggered by BMP-2 and BMP-4 gene transfer showed considerable evidence of hypertrophic differentiation, with, the cells resembling growth plate chondrocytes both morphologically and functionally. This suggests caution when using these candidate genes in cartilage repair."

Red Bone Marrow stem cells(mesenchymal stem cells) such as BMP-2 and BMP-4 can induce hypertrophy of the growth plates

 Potential roles of growth factor PDGF-BB in the bony repair of injured growth plate. 

"Injured growth plate cartilage is often repaired by bony tissue resulting in impaired bone growth in children. Using a rat injury model, our previous studies show that following the injury-induced initial inflammatory response, an influx of mesenchymal-like cells occurs within the growth plate injury site prior to formation of bony tissue. As platelet-derived growth factor (PDGF-BB) is a potent chemotactic factor of mesenchymal cells during skeletal tissue repair, we examined its role during the early fibrogenic response and the subsequent bony repair of injured growth plate. Following growth plate injury, rats received daily injection of the PDGF receptor (PDGFR) inhibitor, Imatinib, for 7 days. Immunohistochemical analysis of injured growth plate at day 1 showed the presence of PDGF-BB expression in some inflammatory cells, while at day 4 PDGFR was expressed by a proportion of the infiltrating mesenchymal cells at the injury site. By day 4, PDGFR inhibition reduced mesenchymal infiltrate (P<0.05); by day 14, Imatinib-treated rats exhibited less bony trabeculae and cartilaginous repair tissues, fewer osteoclasts and less bone marrow (BM) at the injury site, compared to vehicle controls (P<0.01). In vitro "scratch" migration assays with rat BM mesenchymal cells revealed that recombinant PDGF-BB increased cell migration into the "wound" (P<0.05), while Imatinib inhibited this chemotactic response. Quantitative RT-PCR analysis showed that Imatinib treatment decreased expression of the cartilage and bone related genes, Col2a1 and osteocalcin, respectively. These results suggest that PDGF-BB contributes to growth plate injury repair by promoting mesenchymal progenitor cell infiltration, the chondrogenic and osteogenic responses, and remodelling of the repair tissues." 

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

"Injuries to the articular cartilage and growth plate are significant clinical problems due to their limited ability to regenerate themselves. Despite progress in orthopedic surgery and some success in development of chondrocyte transplantation treatment and in early tissue-engineering work, cartilage regeneration using a biological approach still remains a great challenge. In the last 15 years, researchers have made significant advances and tremendous progress in exploring the potentials of mesenchymal stem cells (MSCs) in cartilage repair. These 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. In addition, representing another new perspective for cartilage repair is the successful demonstration of 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. However, despite these rapid pre-clinical advances and some success in engineering cartilage-like tissue and in repairing articular and growth plate cartilage, challenges of their clinical translation remain. To achieve clinical effectiveness, safety, and practicality of using MSCs for cartilage repair, one critical investigation will be to examine 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 mobilizing endogenous MSCs from adjacent source tissues such as synovium, bone marrow, or trabecular bone[This is what lateral synovial joint loading does, it mobilizes the mesenchymal stem cells from the red bone marrow]." 

Are we going to find a study that states exactly what we want?  No.  But what these few studies indicate is that mobilizing endogenous mesencymal stem cells(pushing around red bone marrow stem cells) have a strong possibility to re-activate the growth plates.  The only problem is if you're out of red bone marrow but there's no way of knowing for sure however red bone marrow correlates negatively with age. 

Here's what Hiroki Yokota had to say "I think it possible to hypothesize that this loading[lateral synovial joint loading] re-distributes mesenchymal stem cells." 

Here's a study about overgrowth as a result of fracture.  Now it doesn't distinguish between epiphysis or diasphysis fractures so we don't know which stem cells are causing the overgrowth(red bone marrow or yellow bone marrow): 

Activation of the growth plates on three-phase bone scintigraphy: the explanation for the overgrowth of fractured femurs. 

"Children with an uncomplicated femoral fracture, treated with superimposition of fragments and intentional shortening, usually develop overgrowth of the fractured femur and the ipsilateral tibia which may compensate for the initial shortening and enable the limb in question to reach a length similar to that on the normal side. The overgrowth is evaluated clinically and by scanography. The increased metabolic activity of the growth plates that support this overgrowth has not been documented by any laboratory method. In order to evaluate the metabolic activity of the growth plates, 18 patients (11 males, seven females; mean age 6.1 years) with fractures of the femur were studied at three different time intervals (2-5 months, 6-12 months and 18-24 months). Three-phase bone scintigraphy was performed in all patients. Ten children (five males, five females; mean age 7.5 years) who had had bone imaging for other reasons were used as the control group. Visual analysis of the flow and equilibrium phases was performed for the distal femoral and proximal tibial growth plates. Visual and semi-quantitative analyses of the delayed images were performed for the distal femoral and proximal and distal tibial growth plates. Semi-quantitative analyses yielded the following activity ratios: (a) the distal femoral growth plate of the fractured femur to the contralateral one (FR); (b) the proximal growth plate of the tibia on the side of the fractured femur to the contralateral one (TpR); (c) the distal growth plate of the tibia on the side of the fractured femur to the contralateral one (TdR); and (d) in the control group, the distal growth plates of both femora (FCG) and the proximal (TCGp) and distal (TCGd) growth plates of the tibiae. Visual analysis of the blood flow, equilibrium and delayed images showed increased activity in the distal femoral growth plates during the first and second time intervals[The distal growth plate is the growth plate farthest away from the body, since the body is often in an upright position the bone marrow stem cells should travel downward explaining this phenomena], but not during the third. No significant activity changes were found in the proximal and distal tibial growth plates during any of the phases analysed. The mean and standard deviation for FR in the three time intervals were: FRI=1.22+/-0.27, FRII=1.17+/-0.16 and FRIII=1.09+/-0.20(The bone that was fractured had a faster growth rate). FR values were significantly higher than in the control group (FCG=0.99+/-0.03) (P=0.033). The mean and standard deviation for TpR in the three time intervals were: TpRI=1.08+/-0.18, TpRII=0.94+/-0.09 and TpRIII=0.96+/-0.20. TpR values were not significantly different from those in the control group (TCGp=1.00+/-0.05). However, TpRI was significantly higher than TpRII (P=0.043). The mean and standard deviation for TdR in the three time intervals were: TdRI=1.10+/-0.41, TdRII=1.05+/-0.15 and TdRIII=1.13+/-0.36. TdR values were not significantly higher than in the control group (TCGd=1.00+/-0.04) (P=0.777). These results support the concept that three-phase bone imaging is able to quantify and determine that activation occurs in the distal femoral and proximal tibial growth plates of fractured femora. This phenomenon may explain the overgrowth observed in this injured bone structure." 

The fact that distal growth plates experience overgrowth and not proximal growth plates is consistent with stem cell theory as stem cells should flow downward as the body is often in an upright position.

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