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.
Molecular Pathology Laboratory, Hebrew University-Hadassah Faculty of Dental Medicine, Jerusalem, Israel. email@example.com
Among the approximately 6.5 million fractures suffered in the United States every year, about 15% are difficult to heal. As yet, for most of these difficult cases there is no effective therapy. We have developed a mouse radial segmental defect as a model experimental system for testing the capacity of Genetically Engineered Pluripotent Mesenchymal Cells (GEPMC, C3H10T1/2 clone expressing rhBMP-2), for gene delivery, engraftment, and induction of bone growth in regenerating bone.
Transfected GEPMC expressing rhBMP-2 were further infected with a vector carrying the lacZ gene, that encodes for beta-galactosidase (beta-gal). In vitro levels of rhBMP-2 expression and function were confirmed by immunohistochemistry, and bioassay. Differentiation was assayed using alkaline phosphatase staining. GEPMC were transplanted in vivo into a radial segmental defect. The main control groups included lacZ clones of WT-C3H10T1/2-LacZ, and CHO-rhBMP-2 cells. New bone formation was measured quantitatively via fluorescent labeling, X-ray analysis and histomorphometry. Engrafted mesenchymal cells were localized in vivo by beta-gal expression, and double immunofluorescence.
In vitro, GEPMC expressed rhBMP-2, beta-gal and spontaneously differentiated into osteogenic cells expressing alkaline phosphatase. Detection of transplanted cells revealed engrafted cells that had differentiated into osteoblasts and co-expressed beta-gal and rhBMP-2. Analysis of new bone formation revealed that at four to eight week post-transplantation, GEPMS significantly enhanced segmental defect repair.
Our study shows that cell-mediated gene transfer can be utilized for growth factor delivery to signaling receptors of transplanted cells (autocrine effect) and host mesenchymal cells (paracrine effect) suggesting the ability of GEPMC to engraft, differentiate, and stimulate bone growth. We suggest that our approach should lead to the designing of mesenchymal stem cell based gene therapy strategies for bone lesions as well as other tissues.
- PMID: 10738576 [PubMed – indexed for MEDLINE]
From PubMed study 2 link HERE…
- Mol Ther. 2001 Apr;3(4):449-61.
- Exogenously regulated stem cell-mediated gene therapy for bone regeneration.
Molecular Pathology Laboratory, Hebrew University-Hadassah Medical and Gene Therapy Center, Jerusalem, Israel.
Regulated expression of transgene production and function is of great importance for gene therapy. Such regulation can potentially be used to monitor and control complex biological processes. We report here a regulated stem cell-based system for controlling bone regeneration, utilizing genetically engineered mesenchymal stem cells (MSCs) harboring a tetracycline-regulated expression vector encoding the osteogenic growth factor human BMP-2. We show that doxycycline (a tetracycline analogue) is able to control hBMP-2 expression and thus control MSC osteogenic differentiation both in vitro and in vivo. Following in vivo transplantation of genetically engineered MSCs, doxycycline administration controlled both bone formation and bone regeneration. Moreover, our findings showed increased angiogenesis accompanied by bone formation whenever genetically engineered MSCs were induced to express hBMP-2 in vivo. Thus, our results demonstrate that regulated gene expression in mesenchymal stem cells can be used as a means to control bone healing.
- PMID: 11319905 [PubMed – indexed for MEDLINE] Free full text
From PubMed study 3 link HERE…
- J Gene Med. 2001 May-Jun;3(3):240-51.
- Engineered human mesenchymal stem cells: a novel platform for skeletal cell mediated gene therapy.
Hebrew University-Hadassah Medical and Gene Therapy Center, Jerusalem, Israel.
Human mesenchymal stem cells (hMSCs) are pluripotent cells that can differentiate to various mesenchymal cell types. Recently, a method to isolate hMSCs from bone marrow and expand them in culture was described. Here we report on the use of hMSCs as a platform for gene therapy aimed at bone lesions.
Bone marrow derived hMSCs were expanded in culture and infected with recombinant adenoviral vector encoding the osteogenic factor, human BMP-2. The osteogenic potential of genetically engineered hMSCs was assessed in vitro and in vivo.
Genetically engineered hMSCs displayed enhanced proliferation and osteogenic differentiation in culture. In vivo, transplanted genetically engineered hMSCs were able to engraft and form bone and cartilage in ectopic sites, and regenerate bone defects (non-union fractures) in mice radius bone. Importantly, the same results were obtained with hMSCs isolated from a patient suffering from osteoporosis.
hMSCs represent a novel platform for skeletal gene therapy and the present results suggest that they can be genetically engineered to express desired therapeutic proteins inducing specific differentiation pathways. Moreover, hMSCs obtained from osteoporotic patients can restore their osteogenic activity following human BMP-2 gene transduction, an important finding in the future planning of gene therapy treatment for osteoporosis.
- PMID: 11437329 [PubMed – indexed for MEDLINE]