Arvydas Usas

Synergistic enhancement of bone formation and healing by stem cell–expressed VEGF and bone morphogenetic protein-4

We investigated the interaction between angiogenic and osteogenic factors in bone formation and bone healing with ex vivo gene therapy using muscle-derived stem cells genetically engineered to express human bone morphogenetic protein-4 (BMP4), VEGF, or VEGF-specific antagonist (soluble Flt1). Our results show that although VEGF alone did not improve bone regeneration, it acted synergistically with BMP4 to increase recruitment of mesenchymal stem cells, to enhance cell survival, and to augment cartilage formation in the early stages of endochondral bone formation. These early effects, coupled with accelerated cartilage resorption, eventually led to a significant enhancement of bone formation and bone healing. The beneficial effect of VEGF on bone healing elicited by BMP4 depends critically on the ratio of VEGF to BMP4, with an improper ratio leading to detrimental effects on bone healing. Finally, we show that soluble Flt1 inhibits bone formation elicited by BMP4. Thus, VEGF plays an important role in bone formation elicited by BMP4, and it can significantly enhance BMP4-elicited bone formation and regeneration through multiple mechanisms. This study has important implications for the formulation of new strategies to improve bone healing through increasing mesenchymal stem cell recruitment and survival, in combination with muscle-derived stem cell-based gene therapy.

VEGF Improves, Whereas sFlt1 Inhibits, BMP2‐Induced Bone Formation and Bone Healing Through Modulation of Angiogenesis

We studied the interaction between VEGF and BMP2 during bone formation and bone healing. Results indicate that VEGF antagonist inhibited BMP2‐elicited bone formation, whereas the delivery of exogenous VEGF enhanced BMP2‐induced bone formation and bone healing through modulation of angiogenesis.

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.

Enhancement of tendon-bone integration of anterior cruciate ligament grafts with bone morphogenetic protein-2 gene transfer: a histological and biomechanical study.

Background: The integration of tendon grafts used for replacement of the anterior cruciate ligament is still sometimes unsatisfactory and may be associated with postoperative anterior-posterior laxity. The goal of this study was to examine the capacity of bone morphogenetic protein-2 (BMP-2) gene transfer to improve the integration of semitendinosus tendon grafts at the tendon-bone interface after reconstruction of the anterior cruciate ligament in rabbits. Methods: The anterior cruciate ligaments of adult New Zealand White rabbits were replaced with autologous double-bundle semitendinosus tendon grafts. The semitendinosus tendon grafts had been infected in vitro with adenovirus-luciferase, adenovirus-LacZ (AdLacZ), or adenovirus-BMP-2 (AdBMP-2); untreated grafts served as controls. The grafts were examined histologically at two, four, six, and eight weeks after surgery. In additional experiments, the structural properties of the femur-anterior cruciate ligament graft-tibia complexes, from animals killed eight weeks postoperatively, were determined from uniaxial tests. The stiffness (N/mm) and ultimate load to failure (N) were determined from the resulting load-elongation curves. Results: Genetically engineered semitendinosus tendon grafts expressed reporter genes as well as BMP-2 in vitro. The AdLacZ-infected grafts showed two different histological patterns of transduction. Intra-articularly, infected cells were mostly aligned along the surface, and they decreased in number between two and eight weeks after surgery. In the intra-tunnel portions of the grafts, the number of infected cells did not decrease during the observation period. Moreover, a high number of transduced cells was found in the deeper layers of the tendons. In the control group, granulation-type tissue at the tendon-bone interface showed progressive reorganization into a dense connective tissue, and a later establishment of fibers resembling Sharpey fibers. In the specimens with an AdBMP-2-infected anterior cruciate ligament graft, a broad zone of newly formed matrix resembling chondro-osteoid had formed at the tendon-bone interface at four weeks after surgery. This area was increased at six weeks, showing a transition from bone to mineralized cartilage and nonmineralized fibrocartilage. In addition, in the AdBMP-2-treated specimens, the tendon-bone interface in the osseous tunnel was similar to that of a normal anterior cruciate ligament insertion. The stiffness (29.0 ± 7.1 N/mm compared with 16.7 ± 8.3 N/mm) and the ultimate load to failure (108.8 ± 50.8 N compared with 45.0 ± 18.0 N) were significantly enhanced in the specimens with an AdBMP-2-transduced graft when compared with the control values (p < 0.05). Conclusion: This study demonstrates that BMP-2 gene transfer significantly improves the integration of semitendinosus tendon grafts in bone tunnels after reconstruction of the anterior cruciate ligament in rabbits. Clinical Relevance: Novel technologies including gene therapy and tissue engineering, such as those described in this study, may provide useful therapeutic procedures to enhance biological healing after reconstruction of the anterior cruciate ligament.

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 ¥ 105) 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.

NF-κB inhibition delays DNA damage–induced senescence and aging in mice

The accumulation of cellular damage, including DNA damage, is thought to contribute to aging-related degenerative changes, but how damage drives aging is unknown. XFE progeroid syndrome is a disease of accelerated aging caused by a defect in DNA repair. NF-κB, a transcription factor activated by cellular damage and stress, has increased activity with aging and aging-related chronic diseases. To determine whether NF-κB drives aging in response to the accumulation of spontaneous, endogenous DNA damage, we measured the activation of NF-κB in WT and progeroid model mice. As both WT and progeroid mice aged, NF-κB was activated stochastically in a variety of cell types. Genetic depletion of one allele of the p65 subunit of NF-κB or treatment with a pharmacological inhibitor of the NF-κB-activating kinase, IKK, delayed the age-related symptoms and pathologies of progeroid mice. Additionally, inhibition of NF-κB reduced oxidative DNA damage and stress and delayed cellular senescence. These results indicate that the mechanism by which DNA damage drives aging is due in part to NF-κB activation. IKK/NF-κB inhibitors are sufficient to attenuate this damage and could provide clinical benefit for degenerative changes associated with accelerated aging disorders and normal aging.

Improvement of muscle healing through enhancement of muscle regeneration and prevention of fibrosis

Skeletal muscle is able to repair itself through regeneration. However, an injured muscle often does not fully recover its strength because complete muscle regeneration is hindered by the development of fibrosis. Biological approaches to improve muscle healing by enhancing muscle regeneration and reducing the formation of fibrosis are being investigated. Previously, we have determined that insulin‐like growth factor–1 (IGF‐1) can improve muscle regeneration in injured muscle. We also have investigated the use of an antifibrotic agent, decorin, to reduce muscle fibrosis following injury. The aim of this study was to combine these two therapeutic methods in an attempt to develop a new biological approach to promote efficient healing and recovery of strength after muscle injuries. Our findings indicate that further improvement in the healing of muscle lacerations is attained histologically by the combined administration of IGF‐1 to enhance muscle regeneration and decorin to reduce the formation of fibrosis. This improvement was not associated with improved responses to physiological testing, at least at the time‐points tested in this study. Muscle Nerve 28: 365–372, 2003

Gamma interferon as an antifibrosis agent in skeletal muscle

Muscle injuries are a common problem in sports medicine. Skeletal muscle can regenerate itself, but the process is both slow and incomplete. Previously we and others have used growth factors to improve the regeneration of muscle, but the muscle healing was impeded by scar tissue formation. However, when we blocked the fibrosis process with decorin, an antifibrosis agent, we improved the muscle healing. Here we show that γinterferon (γINF)—a cytokine that inhibits the signaling of transforming growth factor β1 (TGFβ1), a fibrotic stimulator—reduces fibrosis formation and improves the healing of lacerated skeletal muscle. With γINF treatment, the growth rate of muscle‐derived fibroblasts was reduced and the level of fibrotic protein expression induced by TGFβ1 (including TGFβ1, vimentin, and α‐smooth muscle actin) was down‐regulated in vitro. In a mouse laceration model, the area of fibrosis decreased when γINF was injected at either 1 or 2 weeks after injury. More importantly, the injection of γINF at either 1 or 2 weeks post‐injury was found to improve muscle function in terms of both fast‐twitch and tetanic strength. This study demonstrates that γINF is a potent antifibrosis agent that can improve muscle healing after laceration injury.

Enhancement of Bone Healing Based on Ex Vivo Gene Therapy Using Human Muscle-Derived Cells Expressing Bone Morphogenetic Protein 2

Molecular biological advances have allowed the use of gene therapy in a clinical setting. In addition, numerous reports have indicated the existence of inducible osteoprogenitor cells in skeletal muscle. Because of this, we hypothesized that skeletal muscle cells might be ideal vehicles for delivery of bone-inductive factors. Using ex vivo gene transfer methods, we genetically engineered freshly isolated human skeletal muscle cells with adenovirus and retrovirus to express human bone morphogenetic protein 2 (BMP-2). These cells were then implanted into nonhealing bone defects (skull defects) in severe combined immune deficiency (SCID) mice. The closure of the defect was monitored grossly and histologically. Mice that received BMP-2-producing human muscle-derived cells experienced a full closure of the defect by 4 to 8 weeks posttransplantation. Remodeling of the newly formed bone was evident histologically during the 4- to 8-week period. When analyzed by fluorescence in situ hybridization, a small fraction of the transplanted human muscle-derived cells was found within the newly formed bone, where osteocytes normally reside. These results indicate that genetically engineered human muscle-derived cells enhance bone healing primarily by delivering BMP-2, while a small fraction of the cells seems to differentiate into osteogenic cells.

Cartilage repair in a rat model of osteoarthritis through intraarticular transplantation of muscle-derived stem cells expressing bone morphogenetic protein 4 and soluble Flt-1

OBJECTIVE The control of angiogenesis during chondrogenic differentiation is an important issue affecting the use of stem cells in cartilage repair, especially with regard to the persistence of regenerated cartilage. This study was undertaken to investigate the effect of vascular endothelial growth factor (VEGF) stimulation and the blocking of VEGF with its antagonist, soluble Flt-1 (sFlt-1), on the chondrogenesis of skeletal muscle-derived stem cells (MDSCs) in a rat model of osteoarthritis (OA). METHODS We investigated the effect of VEGF on cartilage repair in an immunodeficiency rat model of OA after intraarticular injection of murine MDSCs expressing bone morphogenetic protein 4 (BMP-4) in combination with MDSCs expressing VEGF or sFlt-1. RESULTS In vivo, a combination of sFlt-1- and BMP-4-transduced MDSCs demonstrated better repair without osteophyte formation macroscopically and histologically following OA induction, when compared with the other groups. Higher differentiation/proliferation and lower levels of chondrocyte apoptosis were also observed in sFlt-1- and BMP-4-transduced MDSCs compared with a combination of VEGF- and BMP-4-transduced MDSCs or with BMP-4-transduced MDSCs alone. In vitro experiments with mixed pellet coculture of MDSCs and OA chondrocytes revealed that BMP-4-transduced MDSCs produced the largest pellets, which had the highest gene expression of not only type II collagen and SOX9 but also type X collagen, suggesting formation of hypertrophic chondrocytes. CONCLUSION Our results demonstrate that MDSC-based therapy involving sFlt-1 and BMP-4 repairs articular cartilage in OA mainly by having a beneficial effect on chondrogenesis by the donor and host cells as well as by preventing angiogenesis, which eventually prevents cartilage resorption, resulting in persistent cartilage regeneration and repair.