Biomedical subjects

Johnny Huard

Publications and source records attributed to Johnny Huard.

At least 55 records · Page 3Linked to original sources

PubMed2004

Muscle-derived stem cells.

Researchers have identified 2 types of stem cells in skeletal muscle: satellite cells and multipotent stem cells (MPSCs). The latter category includes different cell populations isolated by various researchers using several techniques. The methods used to isolate these cells appear to influence the stem cell characteristics of the MPSCs. Although MPSCs and satellite cells could represent different stages of maturation of the same progenitor cells, they also could represent distinct populations of stem cells that exist in skeletal muscle. This article summarizes the recent developments in muscle-derived stem cell research.

Animals↗

PubMed2003

Dystrophin delivery in dystrophin-deficient DMDmdx skeletal muscle by isogenic muscle-derived stem cell transplantation.

Duchenne's muscular dystrophy (DMD) is a lethal muscle disease caused by a lack of dystrophin expression at the sarcolemma of muscle fibers. We investigated retroviral vector delivery of dystrophin in dystrophin-deficient DMD(mdx) (hereafter referred to as mdx) mice via an ex vivo approach using mdx muscle-derived stem cells (MDSCs). We generated a retrovirus carrying a functional human mini-dystrophin (RetroDys3999) and used it to stably transduce mdx MDSCs obtained by the preplate technique (MD3999). These MD3999 cells expressed dystrophin and continued to express stem cell markers, including CD34 and Sca-1. MD3999 cells injected into mdx mouse skeletal muscle were able to deliver dystrophin. Though a relatively low number of dystrophin-positive myofibers was generated within the gastrocnemius muscle, these fibers persisted for up to 24 weeks postinjection. The injection of cells from additional MDSC/Dys3999 clones into mdx skeletal muscle resulted in varying numbers of dystrophin-positive myofibers, suggesting a differential regenerating capacity among the clones. At 2 and 4 weeks postinjection, the infiltration of CD4- and CD8-positive lymphocytes and a variety of cytokines was detected within the injected site. These data suggest that the transplantation of retrovirally transduced mdx MDSCs can enable persistent dystrophin restoration in mdx skeletal muscle; however, the differential regenerating capacity observed among the MDSC/Dys3999 clones and the postinjection immune response are potential challenges facing this technology.

Animals↗

PubMed2003

The role of cell type in bone healing mediated by ex vivo gene therapy.

BACKGROUND: The ideal cellular vehicle for use in cell-mediated gene therapy to enhance bone healing has not yet been identified. The purpose of this study was to compare the capacity of two types of cells transduced with retro-bone morphogenetic protein 4 (BMP4)-muscle-derived cells (MDCs) and unfractioned bone marrow stromal cells (BMSCs). METHOD: Primary rat MDCs and unfractioned rat BMSCs were transduced with a retrovirus to express BMP4. A 7-mm, critical-sized femur defect was created in adult rats, and 5 x 10(6) transduced cells were implanted into the femoral defect. Bone healing was monitored radiographically and histologically at 4, 8, and 12 weeks post-implantation. RESULTS: All specimens in the MDC-BMP4 group and BMSC-BMP4 group showed a bridging callus at 8 and 12 weeks. At 12 weeks post-implantation the calluses of the MDC-BMP4 femora displayed significantly higher bone photodensity than the BMSC-BMP4 femora (P<0.05). Histomorphometry revealed no difference between the two treatment groups. However, non-union between newly formed and original bone was observed in none of the MDC femora but in six femora from the BMSC-BMP4 group. CONCLUSION: Both MDCs and unfractioned BMSCs can improve healing of a critical-sized bone defect following transduction of the cells with retroBMP4. However, MDCs appear to yield superior results when compared with BMSCs in terms of improved healing of segmental defects.

Animals↗

PubMed2003

Development of an experimental system for the study of urethral biomechanical function.

Despite its principal mechanical function in the storage and release of urine, the biomechanical properties of the urethra have remained largely unexplored. The purpose of this study was to develop and validate an experimental model that can be used for evaluating whole urethral tissue in such a manner. Bladder-urethral specimens were excised from halothane-anesthetized female rats and mounted at in vivo length within the experimental apparatus consisting of a tissue perfusion chamber, an adjustable fluid column, and a laser micrometer. Outer diameter measurements were made at proximal, mid, and distal axial locations in response to increases in intraluminal pressure and after addition of various muscle-responsive agents. Basal smooth muscle tone and regional variations in compliance were detected through pressure-diameter responses. Chemically evoked contractile responses were measured and correspond to regional compositions of intrinsic smooth and striated muscle components. The results presented illustrate the utility of this system, which should permit a more thorough characterization of structure-function relationships and urethral biomechanical function in relation to normal and dysfunctional tissue states.

Acetylcholine↗

PubMed2003

Gene therapy to improve osteogenesis in bone lesions with severe soft tissue damage.

BACKGROUND: Ex vivo gene therapy can induce bone formation when delivery cells carrying the bone morphogenetic protein (BMP) gene are used. The hypothesis for this study was that the cell-mediated gene therapy could improve the healing of bony lesions with severe soft tissue damage. METHOD: An animal model with a femoral osteotomy lesion associated with soft tissue damage was developed in rats. Muscle-derived cells, genetically engineered to express BMP4, were inserted within the osteotomy gap. Cells genetically engineered to express LacZ were used for the control group. The groups were subdivided with regard to the fixation method: stable and unstable fixation. The rats were killed for histological and radiographic evaluation 3 and 6 weeks post-surgery. RESULTS: No callus formation was found in the control group at any time point, whereas sufficient callus formation appeared in the treatment group after 6 weeks. A bridging callus with woven bone and hypertrophic chondrocytes was achieved in the treatment group when a stable fixation was used, but failed to appear in unstable fixation. CONCLUSION: The combination of muscle-derived cells expressing BMP4 and a stable fixation were able to bridge the bone defect within 6 weeks, but with prolonged osteochondral ossification. Therefore, the ex vivo gene therapy could be an efficient biological approach to improve the treatment of bone lesions with severe soft tissue damage.

Animals↗

PubMed2003

Antifibrotic effects of suramin in injured skeletal muscle after laceration.

Muscle injuries are very common in traumatology and sports medicine. Although muscle tissue can regenerate postinjury, the healing process is slow and often incomplete; complete recovery after skeletal muscle injury is hindered by fibrosis. Our studies have shown that decreased fibrosis could improve muscle healing. Suramin has been found to inhibit transforming growth factor (TGF)-beta1 expression by competitively binding to the growth factor receptor. We conducted a series of tests to determine the antifibrotic effects of suramin on muscle laceration injuries. Our results demonstrate that suramin (50 microg/ml) can effectively decrease fibroblast proliferation and fibrotic-protein expression (alpha-smooth muscle actin) in vitro. In vivo, direct injection of suramin (2.5 mg) into injured murine muscle resulted in effective inhibition of muscle fibrosis and enhanced muscle regeneration, which led to efficient functional muscle recovery. These results support our hypothesis that prevention of fibrosis could enhance muscle regeneration, thereby facilitating more efficient muscle healing. This study could significantly contribute to the development of strategies to promote efficient muscle healing and functional recovery.

3T3 Cells↗

PubMed2003

Muscle-derived stem cells: potential for muscle regeneration.

Duchenne muscular dystrophy (DMD) is a devastating X-linked muscle disease characterized by progressive muscle weakness caused by the lack of dystrophin expression at the sarcolemma of muscle fibers. Although various approaches to delivering dystrophin in dystrophic muscle have been investigated extensively (e.g., cell and gene therapy), there is still no treatment that alleviates the muscle weakness in this common inherited muscle disease. The transplantation of myoblasts can enable transient delivery of dystrophin and improve the strength of injected dystrophic muscle, but this approach has various limitations, including immune rejection, poor cellular survival rates, and the limited spread of the injected cells. The isolation of muscle cells that can overcome these limitations would enhance the success of myoblast transplantation significantly. The efficiency of cell transplantation might be improved through the use of stem cells, which display unique features, including (1) self-renewal with production of progeny, (2) appearance early in development and persistence throughout life, and (3) long-term proliferation and multipotency. For these reasons, the development of muscle stem cells for use in transplantation or gene transfer (ex vivo approach) as treatment for patients with muscle disorders has become more attractive in the past few years. In this paper, we review the current knowledge regarding the isolation and characterization of stem cells isolated from skeletal muscle by highlighting their biological features and their relationship to satellite cells as well as other populations of stem cells derived from other tissues. We also describe the remarkable ability of stem cells to regenerate skeletal muscle and their potential use to alleviate the muscle weakness associated with DMD.

Animals↗

PubMed2003

Gene therapy and tissue engineering for sports medicine.

Sports injuries usually involve tissues that display a limited capacity for healing. The treatment of sports injuries has improved over the past 10 to 20 years through sophisticated rehabilitation programs, novel operative techniques, and advances in the field of biomechanical research. Despite this considerable progress, no optimal solution has been found for treatment of various sports-related injuries, including muscle injuries, ligament and tendon ruptures, central meniscal tears, cartilage lesions, and delayed bone fracture healing. New biological approaches focus on the treatment of these injuries with growth factors to stimulate and hasten the healing process. Gene therapy using the transfer of defined genes encoding therapeutic proteins represents a promising way to efficiently deliver suitable growth factors into the injured tissue. Tissue engineering, which may eventually be combined with gene therapy, may potentially result in the creation of tissues or scaffolds for regeneration of tissue defects following trauma. In this article we will discuss why gene therapy and tissue engineering are becoming increasingly important in modern orthopaedic sports medicine practice. We then will review recent research achievements in the area of gene therapy and tissue engineering for sports-related injuries, and highlight the potential clinical applications of this technology in the treatment of patients with musculoskeletal problems following sports-related injuries.

Athletic Injuries↗

PubMed2003

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.

Animals↗

PubMed2003

Muscle-derived stem cells seeded into acellular scaffolds develop calcium-dependent contractile activity that is modulated by nicotinic receptors.

OBJECTIVES: To explore the contractile activity and physiologic properties of muscle-derived stem cells (MDSCs) incorporated into small intestinal submucosa (SIS) scaffolds. METHODS: MDSCs were harvested from mice hind leg muscles using the preplate technique and stably transfected with a plasmid to express the LacZ reporter gene. Fifty different preparations of SIS cultured with MDSCs (MDSC/SIS) or SIS alone were incubated at 37 degrees C for 1, 4, and 8 weeks and also were mounted in a bath to measure the isometric contractions. RESULTS: LacZ and Masson-trichrome staining revealed MDSCs could migrate into and distribute throughout the SIS and form myotubes. In MDSC/SIS, spontaneous contractile activities were noted in the 4-week (five of six specimens) and 8-week (eight of eight specimens) cultures, but not in 1-week cultures (n = 11). All SIS control groups after 1 (n = 11), 4 (n = 6), and 8 (n = 8) weeks of incubation did not show any activity. In most of the 4-week, and all of the 8-week, MDSC/SIS cultures, the frequency and amplitude of spontaneous contractile activities were decreased by succinylcholine 10 microM and 20 microM. Electrical field stimulation, carbachol, and KCl did not alter the frequency, amplitude, or pattern of spontaneous contractile activities in MDSC/SIS. Spontaneous contractile activities were blocked by Ca(32+)-free Krebs solution with ethyleneglycoltetraacetic acid 200 microM and distilled water. CONCLUSIONS: MDSCs could be incorporated into SIS-forming myotubes capable of contracting. The contractile activity of this three-dimensional construct is Ca(2+) dependent and is modulated by nicotinic receptors. MDSC seeding of an acellular matrix may become a functional sling to reengineer the deficient sphincter or as contractile bladder augmentation.

Animals↗

PubMed2003

Improved sphincter contractility after allogenic muscle-derived progenitor cell injection into the denervated rat urethra.

OBJECTIVES: To study the physiologic outcome of allogenic transplant of muscle-derived progenitor cells (MDPCs) in the denervated female rat urethra. METHODS: MDPCs were isolated from muscle biopsies of normal 6-week-old Sprague-Dawley rats and purified using the preplate technique. Sciatic nerve-transected rats were used as a model of stress urinary incontinence. The experimental group was divided into three subgroups: control, denervated plus 20 microL saline injection, and denervated plus allogenic MDPCs (1 to 1.5 x 10(6) cells) injection. Two weeks after injection, urethral muscle strips were prepared and underwent electrical field stimulation. The pharmacologic effects of d-tubocurare, phentolamine, and tetrodotoxin on the urethral strips were assessed by contractions induced by electrical field stimulation. The urethral tissues also underwent immunohistochemical staining for fast myosin heavy chain and CD4-activated lymphocytes. RESULTS: Urethral denervation resulted in a significant decrease of the maximal fast-twitch muscle contraction amplitude to only 8.77% of the normal urethra and partial impairment of smooth muscle contractility. Injection of MDPCs into the denervated sphincter significantly improved the fast-twitch muscle contraction amplitude to 87.02% of normal animals. Immunohistochemistry revealed a large amount of new skeletal muscle fiber formation at the injection site of the urethra with minimal inflammation. CD4 staining showed minimal lymphocyte infiltration around the MDPC injection sites. CONCLUSIONS: Urethral denervation resulted in near-total abolishment of the skeletal muscle and partial impairment of smooth muscle contractility. Allogenic MDPCs survived 2 weeks in sciatic nerve-transected urethra with minimal inflammation. This is the first report of the restoration of deficient urethral sphincter function through muscle-derived progenitor cell tissue engineering. MDPC-mediated cellular urethral myoplasty warrants additional investigation as a new method to treat stress urinary incontinence.

Animals↗

PubMed2003

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 gammainterferon (gammaINF)--a cytokine that inhibits the signaling of transforming growth factor beta1 (TGFbeta1), a fibrotic stimulator--reduces fibrosis formation and improves the healing of lacerated skeletal muscle. With gammaINF treatment, the growth rate of muscle-derived fibroblasts was reduced and the level of fibrotic protein expression induced by TGFbeta1 (including TGFbeta1, vimentin, and alpha-smooth muscle actin) was down-regulated in vitro. In a mouse laceration model, the area of fibrosis decreased when gammaINF was injected at either 1 or 2 weeks after injury. More importantly, the injection of gammaINF 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 gammaINF is a potent antifibrosis agent that can improve muscle healing after laceration injury.

Animals↗

PubMed2003

Stem cells in the treatment of muscle and connective tissue diseases.

Current data indicate the existence of two types of postnatal stem cells. Tissue non-specific stem cells are haematopoietic in origin and can differentiate into different blood lineages. In contrast, tissue-specific stem cells preferentially differentiate into cells of the residing tissue, although they also possess a limited ability to turn into other lineages. In terms of therapeutic potential, unmodified muscle-derived stem cells have been proven capable of regenerating dystrophic muscle. Furthermore, when genetically modified to express growth factors, these cells are versatile in promoting bone healing. This also occurs with mesenchymal stem cells, which have been used in an attempt to repair defects of cartilage and ligaments. Thus, stem-cell-based therapy--particularly genetically engineered therapy--holds great potential for the treatment of a variety of disorders and conditions affecting the muscle and connective tissue.

Bone and Bones↗

PubMed2003

Muscle stem cells differentiate into haematopoietic lineages but retain myogenic potential.

Muscle-derived stem cells (MDSCs) can differentiate into multiple lineages, including haematopoietic lineages. However, it is unknown whether MDSCs preserve their myogenic potential after differentiation into other lineages. To address this issue, we isolated from dystrophic muscle a population of MDSCs that express stem-cell markers and can differentiate into various lineages. After systemic delivery of three MDSC clones into lethally irradiated mice, we found that differentiation of the donor cells into various lineages of the haematopoietic system resulted in repopulation of the recipients' bone marrow. Donor-derived bone-marrow cells, isolated from these recipients by fluorescence-activated cell sorting (FACS), also repopulated the bone marrow of secondary, lethally irradiated, recipients and differentiated into myogenic cells both in vitro and in vivo in normal mdx mice. These findings demonstrate that MDSC clones retain their myogenic potential after haematopoietic differentiation.

Animals↗

PubMed2003

Gene therapy in orthopaedic surgery.

Gene therapy has the potential to revolutionize the treatment of a variety of musculoskeletal disorders. In the past decade, more than 4,000 patients have been enrolled in clinical trials involving gene therapy. Gene therapy is becoming increasingly important in modern orthopaedic clinical practice. It is important to discuss the issues that physicians and scientists face when designing an experiment or clinical trial involving gene therapy, along with the potential clinical applications for gene therapy in the treatment of patients with musculoskeletal problems.

Bone Development↗

PubMed2002

The role of CD34 expression and cellular fusion in the regeneration capacity of myogenic progenitor cells.

Characterization of myogenic subpopulations has traditionally been performed independently of their functional performance following transplantation. Using the preplate technique, which separates cells based on their variable adhesion characteristics, we investigated the use of cell surface proteins to potentially identify progenitors with enhanced regeneration capabilities. Based on previous studies, we used cell sorting to investigate stem cell antigen-1 (Sca-1) and CD34 expression on myogenic populations with late adhesion characteristics. We compared the regeneration efficiency of these sorted progenitors, as well as those displaying early adhesion characteristics, by quantifying their ability to regenerate skeletal muscle and restore dystrophin following transplantation into allogenic dystrophic host muscle. Identification and utilization of late adhering populations based on CD34 expression led to differential regeneration, with CD34-positive populations exhibiting significant improvements in dystrophin restoration compared with both their CD34-negative counterparts and early adhering cell populations. Regenerative capacity was found to correspond to the level of myogenic commitment, defined by myogenic regulatory factor expression, and the rate and degree of induced cell differentiation and fusion. These results demonstrate the ability to separate definable subpopulations of myogenic progenitors based on CD34 expression and reveal the potential implications of defining myogenic cell behavioral and phenotypic characteristics in relation to their regenerative capacity in vivo.

Animals↗

PubMed2002

Effect of cryoinjury on the contractile parameters of bladder strips: a model of impaired detrusor contractility.

In anesthetized Sprague-Dawley rats, the bladder was exposed and cryoinjury was induced by abruptly freezing the serosal side of the bladder wall with a chilled aluminum rod previously placed on dry ice (-40 degrees C). Five days later, the rats were euthanized, and strips were prepared from the area adjacent to the injury. Neurally and alpha,beta methylene-ATP (alpha,beta m-ATP; 50 microM)-evoked contractions were measured in bladder strips from cryoinjured or intact bladders prepared from sham-operated rats. Cryoinjured bladder strips produced significantly lower contractile forces than intact strips to electrical stimulation at higher (10-40 Hz) frequencies. The maximal rate of the neurally evoked contractions was slower in the cryoinjured bladders. The contractile response to alpha,beta m-ATP was smaller in the cryoinjured preparations indicating that the changes may have also occurred at the postjunctional site. In addition, atropine was more effective at inhibiting the neurally evoked contractions in the cryoinjured bladder strips suggesting that a cholinergic dominance occurs after cryoinjury. It is concluded that cryoinjury is a viable method of causing a defined, reproducible injury to the urinary bladder resulting in impaired function of both the cholinergic transmission and the smooth muscle. The bladder cryoinjury can be used as a model for studying impaired bladder compliance and detrusor contractility as well as treatments that may improve bladder function such as tissue engineering.

Adenosine Triphosphate↗

PubMed2002

Muscle derived cell mediated ex vivo gene transfer to the lower urinary tract: comparison of viral vectors.

Gene therapy is a novel form of molecular medicine that may have a major impact on the future of human health care. We explored the efficacy of skeletal muscle derived cells (MDC) transduced with four viruses for ex vivo gene transfer into the lower urinary tract. Primary MDC were isolated from normal neonatal rats and transduced with: (1). adenovirus, (2). herpes simplex virus type-1 (HSV-1), (3). retrovirus or (4). adeno-associated virus (AAV), all of which express the beta-galactosidase reporter gene. Adult Sprague Dawley rats ( n=4 each group-time) were used. The MDC were injected into the right and left lateral bladder walls. The number of injected MDC ranged from 1 to 1.5 x 10(6). The tissues were harvested after 1, 4, 7, and 15 days, sectioned and assayed for beta-galactosidase expression. In the bladder wall, we noted cells expressing beta-galactosidase for each viral group. Adenoviral and HSV-1 transduced cells showed strong expression at 1 and 4 days post-injection, but the expression decreased gradually and was not detectable at 15 days post-injection. Retroviral transduced cells were detected at each time point with a strong expression persisting for 15 days but decreasing gradually over time. Although expression of the AAV transduced cells was initially weak, the later time points exhibited a much stronger expression, especially at day 7 post-injection. This expression persisted for at least 15 days post-injection. In conclusion, successful MDC mediated ex vivo gene transfer into the lower urinary tract was achieved with all four viral vectors. Our results suggest that the ex vivo approach may lead to an efficient and persistent viral gene delivery to the lower urinary tract while minimizing exposure of the host to virus.

Adenoviridae↗