The effect of platelet-rich plasma on the regenerative therapy of muscle derived stem cells for articular cartilage repair.

OBJECTIVE: Platelet-rich plasma (PRP) is reported to promote collagen synthesis and cell proliferation as well as enhance cartilage repair. Our previous study revealed that the intracapsular injection of muscle derived stem cells (MDSCs) expressing bone morphogenetic protein 4 (BMP-4) combined with soluble Flt-1 (sFlt1) was effective for repairing articular cartilage (AC) after osteoarthritis (OA) induction. The current study was undertaken to investigate whether PRP could further enhance the therapeutic effect of MDSC therapy for the OA treatment. METHODS: MDSCs expressing BMP-4 and sFlt1 were mixed with PRP and injected into the knees of immunodeficient rats with chemically induced OA. Histological assessments were performed 4 and 12 weeks after cell transplantation. Moreover, to elucidate the repair mechanisms, we performed in vitro assays to assess cell proliferation, adhesion, migration and mixed pellet co-culture of MDSCs and OA chondrocytes. RESULTS: The addition of PRP to MDSCs expressing BMP-4 and sFlt1 significantly improved AC repair histologically at week 4 compared to MDSCs expressing BMP-4 and sFlt1 alone. Higher numbers of cells producing type II collagen and lower levels of chondrocyte apoptosis were observed by MDSCs expressing BMP-4 and sFlt1 and mixed with PRP. In the in vitro experiments, the addition of PRP promoted proliferation, adhesion and migration of the MDSCs. During chondrogenic pellet culture, PRP tended to increase the number of type II collagen producing cells and in contrast to the in vivo data, it increased cell apoptosis. CONCLUSIONS: Our findings indicate that PRP can promote the therapeutic potential of MDSCs expressing BMP-4 and sFlt1 for AC repair (4 weeks post-treatment) by promoting collagen synthesis, suppressing chondrocyte apoptosis and finally by enhancing the integration of the transplanted cells in the repair process.

Spine degeneration in a murine model of chronic human tobacco smokers.

OBJECTIVE: To investigate the mechanisms by which chronic tobacco smoking promotes intervertebral disc degeneration (IDD) and vertebral degeneration in mice. METHODS: Three month old C57BL/6 mice were exposed to tobacco smoke by direct inhalation (4 cigarettes/day, 5 days/week for 6 months) to model long-term smoking in humans. Total disc proteoglycan (PG) content [1,9-dimethylmethylene blue (DMMB) assay], aggrecan proteolysis (immunobloting analysis), and cellular senescence (p16INK4a immunohistochemistry) were analyzed. PG and collagen syntheses ((35)S-sulfate and (3)H-proline incorporation, respectively) were measured using disc organotypic culture. Vertebral osteoporosity was measured by micro-computed tomography. RESULTS: Disc PG content of smoke-exposed mice was 63% of unexposed control, while new PG and collagen syntheses were 59% and 41% of those of untreated mice, respectively. Exposure to tobacco smoke dramatically increased metalloproteinase-mediated proteolysis of disc aggrecan within its interglobular domain (IGD). Cellular senescence was elevated two-fold in discs of smoke-exposed mice. Smoke exposure increased vertebral endplate porosity, which closely correlates with IDD in humans. CONCLUSIONS: These findings further support tobacco smoke as a contributor to spinal degeneration. Furthermore, the data provide a novel mechanistic insight, indicating that smoking-induced IDD is a result of both reduced PG synthesis and increased degradation of a key disc extracellular matrix protein, aggrecan. Cleavage of aggrecan IGD is extremely detrimental as this results in the loss of the entire glycosaminoglycan-attachment region of aggrecan, which is vital for attracting water necessary to counteract compressive forces. Our results suggest identification and inhibition of specific metalloproteinases responsible for smoke-induced aggrecanolysis as a potential therapeutic strategy to treat IDD.

Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing.

Several recent studies suggest the isolation of stem cells in skeletal muscle, but the functional properties of these muscle-derived stem cells is still unclear. In the present study, we report the purification of muscle-derived stem cells from the mdx mouse, an animal model for Duchenne muscular dystrophy. We show that enrichment of desmin(+) cells using the preplate technique from mouse primary muscle cell culture also enriches a cell population expressing CD34 and Bcl-2. The CD34(+) cells and Bcl-2(+) cells were found to reside within the basal lamina, where satellite cells are normally found. Clonal isolation and characterization from this CD34(+)Bcl-2(+) enriched population yielded a putative muscle-derived stem cell, mc13, that is capable of differentiating into both myogenic and osteogenic lineage in vitro and in vivo. The mc13 cells are c-kit and CD45 negative and express: desmin, c-met and MNF, three markers expressed in early myogenic progenitors; Flk-1, a mouse homologue of KDR recently identified in humans as a key marker in hematopoietic cells with stem cell-like characteristics; and Sca-1, a marker for both skeletal muscle and hematopoietic stem cells. Intramuscular, and more importantly, intravenous injection of mc13 cells result in muscle regeneration and partial restoration of dystrophin in mdx mice. Transplantation of mc13 cells engineered to secrete osteogenic protein differentiate in osteogenic lineage and accelerate healing of a skull defect in SCID mice. Taken together, these results suggest the isolation of a population of muscle-derived stem cells capable of improving both muscle regeneration and bone healing.

The use of relaxin improves healing in injured muscle.

To improve the functional recovery of injured skeletal muscle, we have focused our efforts on both enhancement of muscle regeneration and prevention of fibrosis. The polypeptide cytokine/growth factor relaxin can inhibit fibrous tissue formation in many tissues. As a member of the insulin-like growth factor family, relaxin also is a potential stimulator of muscle regeneration. In the current experiment, we examined the antifibrotic effect of relaxin in injured skeletal muscle. We also investigated if the injection of relaxin would influence muscle regeneration after injury. Our results demonstrate that relaxin treatment improved histologic and physiologic healing of muscles subjected to traumatic injury.

Effect of bone morphogenetic protein-2-expressing muscle-derived cells on healing of critical-sized bone defects in mice.

BACKGROUND: Cells that express bone morphogenetic protein-2 (BMP-2) can now be prepared by transduction with adenovirus containing BMP-2 cDNA. Skeletal muscle tissue contains cells that differentiate into osteoblasts on stimulation with BMP-2. The objectives of this study were to prepare BMP-2-expressing muscle-derived cells by transduction of these cells with an adenovirus containing BMP-2 cDNA and to determine whether the BMP-2-expressing muscle-derived cells would elicit the healing of critical-sized bone defects in mice. METHODS: Primary cultures of muscle-derived cells from a normal male mouse were transduced with adenovirus encoding the recombinant human BMP-2 gene (adBMP-2). These cells (5 yen 10(5)) were implanted into a 5-mm-diameter critical-sized skull defect in female SCID (severe combined immunodeficiency strain) mice with use of a collagen sponge as a scaffold. Healing in the treatment and control groups was examined grossly and histologically at two and four weeks. Implanted cells were identified in vivo with use of the Y-chromosome-specific fluorescent in situ hybridization (FISH) technique, and their differentiation into osteogenic cells was demonstrated by osteocalcin immunohistochemistry. RESULTS: Skull defects treated with muscle cells that had been genetically engineered to express BMP-2 had >85% closure within two weeks and 95% to 100% closure within four weeks. Control groups in which the defect was not treated (group 1), treated with collagen only (group 2), or treated with collagen and muscle cells without adBMP-2 (group 3) showed at most 30% to 40% closure of the defect by four weeks, and the majority of the skull defects in those groups showed no healing. Analysis of injected cells in group 4, with the Y-chromosome-specific FISH technique showed that the majority of the transplanted cells were located on the surfaces of the newly formed bone, but a small fraction (approximately 5%) was identified within the osteocyte lacunae of the new bone. Implanted cells found in the new bone stained immunohistochemically for osteocalcin, indicating that they had differentiated in vivo into osteogenic cells. CONCLUSIONS: This study demonstrates that cells derived from muscle tissue that have been genetically engineered to express BMP-2 elicit the healing of critical-sized skull defects in mice. The cells derived from muscle tissue appear to enhance bone-healing by differentiating into osteoblasts in vivo. CLINICAL RELEVANCE: Ex vivo gene therapy with muscle-derived cells that have been genetically engineered to express BMP-2 may be used to treat nonhealing bone defects. In addition, muscle-derived cells appear to include stem cells, which are easily obtained with muscle biopsy and could be used in gene therapy to deliver BMP-2.

New use of a three-dimensional pellet culture system for human intervertebral disc cells: initial characterization and potential use for tissue engineering.

STUDY DESIGN: Human intervertebral disc cells were cultured in a new three-dimensional "pellet culture" system as an alternative to conventional alginate bead microspheres. Histologic, biochemical, and immunohistologic assays were performed to characterize this new culturing method for disc cells. The feasibility of using the pellet culture system to study effects of gene therapy was also assessed. OBJECTIVES: To characterize a new and simpler, three-dimensional culture system for human intervertebral disc cells and to assess the feasibility of its use for gene therapy and tissue engineering studies. SUMMARY OF BACKGROUND DATA: The alginate microsphere three-dimensional culture system has been the most utilized culture method for disc cells, but it is technically difficult and has some disadvantages. Recently, the "pellet culture" method, a simpler three-dimensional culture system, was described for bone marrow stromal cells. This simpler method might be useful for in vitro and in vivo studies of disc cells as well as for delivery of exogenous genes. METHODS: Isolated human intervertebral disc cells were centrifuged at low speeds to form aggregates and allowed to grow as pellets for up to 3 weeks. At various times these pellet cultures were analyzed grossly, histologically, and immunohistologically. Their ability to incorporate [35S]sulfate in their response to TGF-beta1 was also analyzed. The ability of these pellets to deliver and express exogenous genes in vivo was analyzed by implantation of pellet cultures in muscles of SCID mice. RESULTS: Within several days the intervertebral disc cells were able to form mature three-dimensional aggregates that were each well encapsulated by a fibrous capsule. These pellets successfully synthesized proteoglycan and collagen Type II matrix as determined by histology and immunohistochemistry. In response to TGF-beta1, the pellets increased synthesis of proteoglycan and collagen Type II. When implanted into thigh muscles of SCID mice, the pellets remained aggregated and expressed the beta-galactosidase marker gene in vivo for up to 2 weeks. CONCLUSIONS: The pellet culture system is a new and technically simple method to culture human intervertebral disc cells. The majority of the human intervertebral disc cells retained their native phenotype in this three dimensional system and expressed a marker gene both in vitro and in vivo. Thus, this pellet system might be useful for in vitro and in vivo biochemical studies as well as for studies involving gene therapy and tissue engineering.

The use of an antifibrosis agent to improve muscle recovery after laceration.

Muscle injuries are challenging problems in traumatology and the most frequent injuries in sports medicine. Muscle injuries are capable of healing, although slowly and occasionally with incomplete functional recovery. We observed that lacerated muscle undergoes a rapid process of regeneration, which is hindered by the development of fibrosis. Biologic approaches to enhance muscle regeneration and prevent fibrosis are being investigated to improve muscle healing after injuries. We observed that growth factors can improve muscle regeneration but cannot prevent muscle fibrosis. We investigated the use of an antifibrosis substance, decorin, as an approach to prevent fibrosis and thereby improve muscle healing after injury in murine muscle. We observed that direct injection of human recombinant decorin can efficiently prevent fibrosis and enhance muscle regeneration in the lacerated muscle. More importantly, decorin can improve the recovery of strength in the injured muscle to a level similar to that observed in normal noninjured muscle. These results suggest that injection of decorin improves both the muscle structure and the function of the lacerated muscle to near complete recovery. This study will contribute significantly to the development of strategies to promote efficient muscle healing and complete functional recovery after muscle injuries.

[Ex-vivo gene therapy with BMP-4 for critically sized defects and enhancement of fracture healing in an osteoporotic animal model]. / Ex-vivo-Gentherapie mit BMP4 bei kritischen Substanzdefekten und Frakturen im osteoporotischen Tiermodell.

Fractures in osteoporotic bones or segment defects are problematic bone lesions with a reduced biological capability of regeneration. We tested the hypothesis that cell-mediated ex vivo gene therapy to deliver BMP4 can heal critically sized defects and improve bone healing in osteoporotic rats. Primary muscle-derived cells were isolated from the hindlimb muscle of rats and retrovirally transduced to express bone morphogenic protein 4 (BMP4). The bone formation was evaluated following local application of these cells in critically sized defects and in fractures of osteoporotic bones. Radiographic analysis revealed bridging callus formation in a critically sized defect in all specimens using muscle-derived cells expressing BMP4 at 12 weeks. These findings were confirmed by histological evaluation, which revealed callus bone formation with good integration to the distal and proximal bone. Following treatment with muscle-derived cells expressing BMP4, the bone healing process in the osteoporotic bone was improved to the level similar to that of normal bone. The ex vivo gene therapy could be a promising tool for the treatment of osteoporotic fractures and critically sized defects. The reduced number of complications (nonunions, loss of reduction, and fragment dislocation), shortening of hospitalization period, and improvement of bone strength are decisive advocates for this treatment option.

Analysis of rabbit articular cartilage repair after chondrocyte implantation using optical coherence tomography.

OBJECTIVE: To evaluate the utility and limitations of optical coherence tomography (OCT) for immediate, high-resolution structural analysis of rabbit articular repair tissue following chondrocyte implantation without excising or sectioning the specimen. METHODS: Full thickness articular cartilage defects were created in the patellar grooves of 30 adult rabbit knee joints. Allogenic cultured chondrocytes embedded in collagen gels were implanted into the surgical defects. A periosteal patch was then sutured over the chondrocyte-collagen composites. Six animals per time point were sacrificed at 2, 4, 8, 12 and 24 weeks after surgery. The repair tissues were sequentially analysed by arthroscopic surface imaging, OCT, and histology. The resulting images were compared to determine qualitative and quantitative features of surface roughness, repair tissue integration, and micro-architecture. Statistical analysis was performed using Student's t -testing and linear regression. RESULTS: OCT was able to identify the bone and cartilage interface in normal rabbit articular cartilage and regenerated cartilage at 24 weeks post chondrocyte implantation. OCT was able to identify hypertrophy at 4 and 8 weeks, and subtle surface fibrillations at 24 weeks, comparable with histological analysis at low magnification (20x). More importantly, OCT was able to detect embedded gaps between the repair tissue and surrounding host cartilage. CONCLUSION: Close correlation was observed between OCT and histological analysis of morphological features important to the assessment of articular cartilage repair. These results demonstrate that OCT is capable of providing immediate 'optical biopsy' of the rabbit articular cartilage repair tissue without damaging the specimen, and suggest that this new technique, if integrated with an arthroscope, can potentially be used in longitudinal studies of articular cartilage repair in vivo.