Polyurethane Scaffolds Seeded With Genetically Engineered Skeletal Myoblasts: A Promising Tool to Regenerate Myocardial Function

In animal models, intramyocardial injection of primary skeletal myoblasts is supposed to promote tissue regeneration and to improve cardiac function after myocardial infarction. The usage of genetically engineered myoblasts overexpressing the paracrine factors involved in tissue repair is believed to enhance these effects. However, cell therapy via injection is always accompanied by a high death rate of the injected cells. Here, we describe the construction of a growth factor‐producing myoblast‐seeded scaffold to overcome this limitation. Skeletal myoblasts were isolated and expanded from newborn Lewis rats. Cells were seeded on polyurethane (PU) scaffolds (Artelon) and transfected with DNA of VEGF‐A, HGF, SDF‐1, or Akt1 using the lipid‐based Metafectene Pro method. Overexpression was verified by ELISA, RT‐PCR (VEGF‐A, HGF, and SDF‐1) and Western blot analysis (Akt1). The seeded scaffolds were transplanted onto damaged myocardium of Lewis rats 2 weeks after myocardial infarction. Six weeks later, their therapeutic potential in vivo was analyzed by measurement of infarction size and capillary density. Primary rat skeletal myoblasts seeded on PU scaffolds were efficiently transfected, achieving transfection rates of 20%. In vitro, we noted a significant increase in expression of VEGF‐A, HGF, SDF‐1, and Akt1 after transfection. In vivo, transplantation of growth factor‐producing myoblast‐seeded scaffolds resulted in enhanced angiogenesis (VEGF‐A, HGF, and Akt1) or a reduced infarction zone (SDF‐1 and Akt1) in the ischemically damaged myocardium. In summary, we constructed a growth factor‐producing myoblast‐seeded scaffold which combines the beneficial potential of stem cell transplantation with the promising effects of gene‐therapeutic approaches. Because this matrix also allows us to circumvent previous cell application drawbacks, it may represent a promising tool for tissue regeneration and the re‐establishment of cardiac function after myocardial infarction.     

Enhancement of the functional benefits of skeletal myoblast transplantation by means of coadministration of hypoxia-inducible factor 1alpha

OBJECTIVE: Early cell death remains a major limitation of skeletal myoblast transplantation. Because the poor vascularization of the target scars contributes to cell loss, we assessed the effects of combining skeletal myoblast transplantation with administration of hypoxia-inducible factor 1alpha, a master gene that controls the expression of a wide array of angiogenic factors. METHODS: A myocardial infarction was created in 56 rats by means of coronary artery ligation. Eight days later, rats were randomly allocated to receive in-scar injections of culture medium (control animals, n = 11), skeletal myoblasts (5 x 10(6) , n = 13), adenovirus-encoded hypoxia-inducible factor 1alpha (1.0 x 10(10) pfu/mL, n = 7), or skeletal myoblasts (5 x 10(6)) in combination with an empty vector (n = 3) or active hypoxia-inducible factor 1alpha (1.0 x 10(10) pfu/mL, n = 13). A fifth group (n = 9) underwent a staged approach in which hypoxia-inducible factor 1alpha (1.0 x 10(10) pfu/mL) was injected at the time of infarction, followed 8 days later by skeletal myoblasts (5 x 10(6)). Left ventricular function was assessed echocardiographically before transplantation and 1 month thereafter. Explanted hearts were then processed for the immunohistochemical detection of myotubes, quantification of angiogenesis, myoblast engraftment, and cell survival. RESULTS: Baseline ejection fractions were not significantly different among groups (35%-40%). One month later, ejection fraction had decreased from baseline in control hearts and in those injected with hypoxia-inducible factor 1alpha. In contrast, it did not deteriorate after injections of skeletal myoblasts alone or combined with either the empty vector or active hypoxia-inducible factor 1alpha administered sequentially. The most striking change occurred in the skeletal myoblast plus hypoxia-inducible factor 1alpha combined group in which ejection fraction increased dramatically (by 27%) above baseline levels and was thus markedly higher than in all other groups ( P = .0001 and P = .001 vs control animals and animals receiving hypoxia-inducible factor 1alpha, respectively). Compared with skeletal myoblasts alone, the coadministration of hypoxia-inducible factor 1alpha resulted in a significantly greater degree of angiogenesis, cell engraftment, and cell survival. CONCLUSION: Induction of angiogenesis is an effective means of potentiating the functional benefits of myoblast transplantation, and hypoxia-inducible factor 1alpha can successfully achieve this goal.     

Can erythropoietin improve skeletal myoblast engraftment in infarcted myocardium?

The benefits of skeletal myoblast transplantation are limited by the high rate of early cell death which is partly of ischemic origin. We, therefore, assessed whether graft survival could be improved by the additional use of the angiogenic cytokine erythropoietin (EPO). Thirty-five Lewis rats underwent coronary artery ligation and, two weeks later, were randomized to receive in-scar injections of control medium, skeletal myoblasts (5x10(6)) or skeletal myoblasts with EPO started the day before transplantation and continued for two weeks (500 U/kg three times a week). A fourth group was treated by EPO alone without injections. Function was assessed by 2D echocardiography before transplantation and one month thereafter. Compared with controls and hearts treated by EPO-alone, those transplanted with myoblasts yielded a significantly better recovery of LV ejection fraction, irrespective of whether they had received EPO or not. Neither the area of myoblast engraftment, nor angiogenesis differed between the myoblast-alone and the myoblast+EPO groups. Apoptosis was hardly detectable and, therefore, unaffected by EPO therapy. In this model, EPO failed to improve myoblast engraftment and postinfarction LV function. These negative findings justify to pursue the search for alternate cell survival-enhancing strategies.     

Long‐Term Evaluation of Myoblast Seeded Patches Implanted on Infarcted Rat Hearts

Cell transplantation presents great potential for treatment of patients with severe heart failure. However, its clinical application was revealed to be more challenging than initially expected in experimental studies. Further investigations need to be undertaken to define the optimal treatment conditions. We previously reported on the epicardial implantation of a bio‐engineered construct of skeletal myoblast‐seeded polyurethane and its preventive effect on progression toward heart failure. In the present study, we present a long‐term evaluation of this functional outcome. Left anterior descending coronary ligation was performed in female Lewis rats. Two weeks later, animals were treated with either epicardial implantation of biograft, acellular scaffold, sham operation, or direct intramyocardial skeletal myoblast injection. Functional assessments were performed with serial echocardiographies every 3 months and end point left ventricle pressure was assessed. Hearts were then harvested for histological examinations. Myocardial infarction induced a slow and progressive reduction in fractional shortening after 3 months. Progression toward heart failure was significantly prevented for up to 6 months after injection of myoblasts and for up to 9 months following biograft implantation. Nevertheless, this effect vanished after 12 months, with immunohistological examinations revealing an absence of the transplanted myoblasts within the scaffold. We demonstrated that tissue therapy is superior to cell therapy for stabilization of heart function. However, beneficial effects are transient.     

Hepatocyte growth factor-transfected skeletal myoblasts to limit the development of postinfarction heart failure

Stem cells transplanted to an injured heart affect the host myocardium indirectly. The cytokine hepatocyte growth factor (HGF) may play a key role in this paracrine activity. We hypothesized that HGF‐overexpressing stem cells would restore cardiac function after myocardial infarction (MI). Because there is a high rate of cell death when injecting the cells intramyocardially, we used scaffold‐based cell transfer. Skeletal myoblasts (SkMs) were isolated and expanded from newborn Lewis rats. Cells were transfected with pcDNA3‐huHGF and seeded on polyurethane (PU) scaffolds or diluted in medium for cell injection. The seeded scaffolds were transplanted in rats two weeks after MI (group: PU‐HGF‐SkM) or the infection solution was intramyocardially injected (group: Inj‐HGF‐SkM). Two groups (Inj‐SkM and PU‐SkM) have been prepared with untransfected cells and sham group without any cell therapy served as control (n = 10 each group). At the beginning of treatment (baseline) and six weeks later, hemodynamic parameters were assessed. At the end of the study, histological analysis was employed. In sham animals we detected a decrease in systolic and diastolic function during the observation time. Treatment with untransfected myoblasts did not lead to any significant changes in hemodynamic parameters between the intervention and six weeks later. In group PU‐HGF‐SkM, systolic parameters like dP/dt(max), dP/dt(min) and isovolumic contraction improved significantly from baseline to study end. Some diastolic parameters were inferior as compared to baseline (SB‐Ked, pressure half time [PHT], Tau). In group Inj‐HGF‐SkM, only PHT was impaired as compared to preinterventional values. Histological analysis showed significantly more capillaries in the infarction border zone in groups PU‐HGF‐SkM than in sham and Inj‐SkM group. The infarction size was not affected by the therapy. Transplanting HGF‐transfected myoblasts after MI can limit the development of ventricular dysfunction. Scaffold‐based therapy in combination with gene therapy accelerates this capacity. This hemodynamic amelioration is accompanied by neovascularization, but not by smaller infarction sizes.     

Effectiveness of transient immunosuppression using cyclosporine for xenomyoblast transplantation for cardiac repair

We studied the survival of human myoblast for cellular myocardial reconstruction in a porcine model of chronic myocardial ischemia with immune tolerance using transient immunosuppression. A porcine model of chronic cardiac ischemia was created in 10 pigs (DMEM medium-injected n = 4; myoblast transplanted n = 6) by clamping ameroid ring around left circumflex coronary artery. Three weeks later, 3 x 10(8) human myoblasts carrying lac-z reporter gene were transplanted in multiple sites (0.25 mL each) into the left ventricular wall. Immunosuppression was achieved with 5 mg/kg cyclosporine for 6 weeks after cell transplantation. After animals were euthanized between 6 and 30 weeks after cell transplantation; the heart was removed for histological studies. Discontinuation of immunosuppression after 6 weeks of cell transplantation did not result in donor cell rejection. The lac-z-positive donor cells were detected in porcine host cardiac tissue for up to 30 weeks posttransplantation, expressing human skeletal myosin heavy chain. The results highlight the effectiveness of transient immunosuppression for myoblast transplantation for cardiac repair.     

Combined autologous cellular cardiomyoplasty with skeletal myoblasts and bone marrow cells in canine hearts for ischemic cardiomyopathy

OBJECTIVES: Cellular cardiomyoplasty with isolated skeletal myoblasts and bone marrow mononuclear cells is an encouraging therapeutic strategy for heart failure. We investigated the achievements accomplished with combined cell therapy of skeletal myoblast and bone marrow mononuclear cell transplantation to the ischemic canine myocardium. METHODS: Autologous skeletal myoblasts (1 x 10(8)) and autologous bone marrow mononuclear cells (3 x 10(6)) were injected directly into the damaged myocardium of canine hearts that had undergone 2 weeks of left anterior descending coronary artery ligation. Treatment groups were as follows: skeletal myoblasts plus bone marrow mononuclear cells (combined cell therapy, n = 4), myoblasts (n = 4), bone marrow mononuclear cells (n = 4), and medium only (n = 4). In similarly designed supporting experiments, angiogenic factor expression was evaluated by enzyme-linked immunosorbent assay after cell transplantation in rat hearts that had undergone left anterior descending coronary artery ligation. RESULTS: Four weeks after cell implantation, echocardiography demonstrated better cardiac performance with reduced left ventricular dilation and significantly improved ejection fraction in the combined cell therapy group compared with that seen in the other groups (pretreatment, 37.7% +/- 1.1%, vs combined cell therapy, 55.4% +/- 8.6%; myoblasts, 47.4% +/- 7.4%; bone marrow mononuclear cells, 44.4% +/- 6.7%; medium only [control], 34.4% +/- 5.4%; P < .05). A significantly high number of neovessels were observed in the group receiving combined cell therapy only (combined cell therapy, 45.5 +/- 12 x 10(2)/mm2; myoblasts, 26.5 +/- 8 x 10(2)/mm2; bone marrow mononuclear cells, 30.7 +/- 15 x 10(2)/mm2; medium only [control], 7.1 +/- 1 x 10(2)/mm2; P < .05). Immunostained sections expressed the skeletal specific marker myosin heavy chain, although they did not express the cardiac specific marker troponin T. Results of enzyme-linked immunosorbent assay showed the highest expression of vascular endothelial growth factor (combined cell therapy, 2.9 +/- 0.7 ng/g tissue; myoblasts, 0.24 +/- 0.7 ng/g tissue; bone marrow mononuclear cells, 1.9 +/- 0.2 ng/g tissue; medium only [control], 0.19 +/- 0.004 ng/g tissue; P < .05) and hepatocyte growth factor in the combined cell therapy hearts. CONCLUSIONS: Combined autologous cellular therapy induced both myogenesis and angiogenesis with enhancement of cardiac performance and reduction of cardiac remodeling, suggesting a capable strategy for treating severe ischemic cardiomyopathy clinically.     

小肠黏膜下层复合上皮细胞及成肌细胞构建组织工程食管的初步研究

目的初步探索利用小肠黏膜下层(smallintestinalsubmucosa,SIS)作为支架材料,复合食管鳞状上皮细胞与成肌细胞构建组织工程食管的可行性。方法4周龄无胸腺小鼠20只,体重20.0±2.5g,雌雄不限。体外分离、培养人胚胎食管鳞状上皮细胞、成肌细胞,并行5-BrdU标记;制备SIS材料并切割成1cm×1cm大小,在SIS材料同一表面接种两种细胞,待细胞在材料表面贴附后将其植入裸鼠体内深筋膜下,于术后第3天及1、2、3周取材行组织学和抗角蛋白、平滑肌肌动蛋白(smoothmuscleactin,SMA)免疫组织化学观察,以了解食管鳞状上皮细胞、成肌细胞在SIS材料上的增殖、分化情况和复合物在裸鼠体内的血管化情况。结果人胚胎食管鳞状上皮细胞、成肌细胞能够渗透、生长于SIS材料中,并形成数层结构。植入体内第3天可见细胞增殖为多层覆盖于材料表面,并分泌大量细胞间基质。第2周时已经形成7~8层细胞,并伴有血管长入。3周时细胞增殖为十几层,大量血管长入。通过5-BrdU标记抗体染色观察,示SIS支架材料上生长的细胞多为所植入细胞。抗角蛋白,SMA免疫组织化学染色示移植细胞能够在体内分化。结论成肌细胞与鳞状上皮细胞在SIS材料上的复合培养物,在体内能继续增殖分化形成多层细胞结构,并能快速血管化,可用于组织工程化食管的构建。     

A role for natural killer cells in the rapid death of cultured donor myoblasts after transplantation

BACKGROUND: The rapid and massive death of cultured donor myoblasts after injection in vivo is a major problem for clinical myoblast transfer therapy (MTT). This study shows blood-borne factors are responsible and that ablating the host natural killer (NK) cell response greatly enhances the survival of such donor myoblasts. METHODS: Cultured male donor myoblasts were injected into muscles of female host mice and surviving donor male DNA (myoblasts) quantified using a Y-chromosome specific (Y1) probe. Survival of donor myoblasts transfected with m144, a murine major histocompatibility complex (MHC) class I homologue that protects against NK attack, was quantified. In addition, donor myoblast survival was investigated in host mice following initial (before MTT) and sustained (repeatedly for 3 weeks after MTT) depletion of host NK1.1+ and CD4+/CD8+ cells using specific monoclonal antibodies (either alone or in combination) for up to 3 weeks after MTT, as well as in beige (deficient in NK activity) and in perforin-deficient mdx host mice. RESULTS: A major role for blood-borne factors (especially cells) was confirmed by MTT experiments in irradiated and perfused host mice. Substantially enhanced myoblast survival was seen with donor myoblasts modified by transfection with the m144 molecule or following antibody depletion of host NK1.1+ cells and in beige host mice. Other studies support some role for CD8+ but not CD4+ cells. CONCLUSIONS: These combined data support a central role for host NK cells in the rapid initial death of donor myoblasts. The demonstrated role of NK cells provides strategies to enhance the efficacy of clinical myoblast transplantation.     

Overexpression of connexin 43 in skeletal myoblasts: Relevance to cell transplantation to the heart

OBJECTIVE: Skeletal myoblast transplantation is a promising strategy for treating end-stage heart failure. One potential problem in the development of functional, synchronously contracting grafts is the degree of intercellular communication between grafted myoblasts and host cardiomyocytes. Thus it is expected that enhancement of intercellular gap junction formation would result in improved efficiency of skeletal myoblast transplantation. In this study we investigated whether myoblasts overexpressing connexin 43, a major cardiac gap junction protein, would enhance this intercellular communication. METHODS AND RESULTS: L6 rat skeletal myoblast cell lines overexpressing connexin 43 were generated by means of gene transfection and clonal selection. Connexin 43 overexpression of these myoblasts, which continued both in undifferentiated and differentiated states (up to 17-fold greater protein level in comparison with control-transfected myoblasts, as measured with Western blotting), was observed on cell surfaces where gap junctions should exist. Both dye microinjection and scrape loading with fluorescent dyes showed enhancement in intercellular dye transfer between connexin 43-transfected myoblasts compared with that found in control-transfected cells. Morphologically, these myoblasts fused and differentiated into multinucleated myotubes more rapidly, demonstrating a higher level of cellular creatine kinase activity as a marker of myogenic differentiation throughout the culture period compared with that of control-transfected myoblasts. CONCLUSIONS: We have generated connexin 43-overexpressing skeletal myoblast cell lines that resulted in improved formation of functional intercellular gap junctions, which could be relevant to synchronous contraction of grafted myoblasts in the heart. In addition, these cells demonstrated more rapid differentiation, which would also be advantageous in a graft for transplantation to the heart.     

Transplantation of cryopreserved muscle cells in dilated cardiomyopathy: effects on left ventricular geometry and function

OBJECTIVE: Cell transplantation to prevent congestive heart failure in patients with inherited dilated cardiomyopathy might require the use of noncardiac donor cells unaffected by the genetic defect and cryopreservation to permit cell storage until the time of transplantation. However, the effects of cryopreservation on peripheral muscle cells harvested from a cardiomyopathic recipient and their subsequent ability to restore cardiac structure and function after transplantation are unknown. METHODS: Skeletal myoblasts and vascular smooth muscle cells from cardiomyopathic hamsters (delta-sarcoglycan-deficient BIO 53.58 hamster) and age-matched normal donor hamsters were isolated, expanded in culture, and cryopreserved. After reanimation in culture, cell morphology and growth rate were assessed and compared with values seen in noncryopreserved cells. A total of 4 x 10(6) previously cryopreserved skeletal myoblasts (n = 10) and vascular smooth muscle cells (n = 10) harvested from cardiomyopathic donors were then transplanted into the left ventricles of 17-week-old BIO 53.58 hamsters. Hearts injected with culture medium alone (n = 11) served as controls. Heart function was assessed 5 weeks after transplantation on a Langendorff apparatus, and left ventricular geometry was quantified by means of computerized planimetry. Staining with 5-bromo-2'-deoxyuridine identified the injected cells. RESULTS: Vascular smooth muscle cells from cardiomyopathic donors had an abnormal morphology and diminished growth rates in culture compared with vascular smooth muscle cells from normal donors. These markers of injury were exacerbated by cryopreservation. In contrast, vascular smooth muscle cells from normal donors and skeletal myoblasts from either cardiomyopathic or normal donors appeared normal in culture and were unaffected by cryopreservation. Both cryopreserved vascular smooth muscle cells and skeletal myoblasts from cardiomyopathic donors formed a viable muscle-resembling tissue that prevented wall thinning, limited left ventricular dilatation, and preserved global systolic function in hamsters with a genetic dilated cardiomyopathy. However, attenuation of cardiac remodeling and preservation of global heart function was greater after skeletal myoblast transplantation compared with vascular smooth muscle cell transplantation in parallel to the in vitro morphologic and growth characteristics of these cells. CONCLUSIONS: Cryostorage of healthy donor cells does not prevent the benefits of cell transplantation on limiting remodeling and preserving cardiac function in the failing heart. The health of donor cells in vitro predicts their subsequent benefits on cardiac structure and function after transplantation. Cryopreservation of donor cells might facilitate a clinically applicable and effective approach for ventricular restoration with cell-transplantation therapy for patients with inherited dilated cardiomyopathy.     

Comparison of the effects of fetal cardiomyocyte and skeletal myoblast transplantation on postinfarction left ventricular function

OBJECTIVES: Transplantation of fetal cardiomyocytes improves function of infarcted myocardium but raises availability, immunologic, and ethical issues that justify the investigation of alternate cell types, among which skeletal myoblasts are attractive candidates. METHODS: Myocardial infarction was created in rats by means of coronary artery ligation. One week later, the animals were reoperated on and intramyocardially injected with culture growth medium alone (controls, n = 15), fetal cardiomyocytes (5 x 10(6) cells, n = 11), or neonatal skeletal myoblasts (5 x 10(6) cells, n = 16). The injections consisted of a 150-microL volume and were made in the core of the infarct, and the animals were immunosuppressed. Left ventricular function was assessed by echocardiography immediately before transplantation and 1 month thereafter. Myoblast-transplanted hearts were then immunohistologically processed for the expression of skeletal muscle-specific embryonic myosin heavy chain and cardiac-specific connexin 43. RESULTS: The left ventricular ejection fraction markedly increased in the fetal and myoblast groups from 39.3% +/- 3.9% to 45% +/- 3.4% (P =.086) and from 40.4% +/- 3.6% to 47.3% +/- 4.4% (P =.034), respectively, whereas it decreased in untreated animals from 40.6% +/- 4% to 36.7% +/- 2.7%. Transplanted myoblasts could be identified in all animals by the positive staining for skeletal muscle myosin. Conversely, clusters of connexin 43 were not observed on these skeletal muscle cells. CONCLUSIONS: These results support the hypothesis that skeletal myoblasts are as effective as fetal cardiomyocytes for improving postinfarction left ventricular function. The clinical relevance of these findings is based on the possibility for skeletal myoblasts to be harvested from the patient himself.     

Feasibility and safety of autologous myoblast transplantation in patients with ischemic cardiomyopathy

Successful autologous skeletal myoblast transplantation into infarcted myocardium in a variety of animal models has demonstrated improvement in cardiac function. We evaluated the safety and feasibility of transplanting autologous myoblasts into infarcted myocardium of patients undergoing concurrent coronary artery bypass grafting (CABG) or left ventricular assist device implantation (LVAD). In addition, we sought to gain preliminary information on graft survival and any potential improvement of cardiac function. Eighteen patients with a history of ischemic cardiomyopathy participated in a phase I, nonrandomized, multicenter pilot study of autologous skeletal myoblast transplantation concurrent with CABG or LVAD implantation. Twelve patients with a history of previous myocardial infarction (MI) and a left ventricular ejection of less than 30% were enrolled in the CABG arm. In a second arm, six patients underwent LVAD implantation as a bridge to heart transplantation and were required to donate their heart for testing at the time of heart transplant. Myoblasts were successfully transplanted in all patients without any acute injection-related complications or significant long-term unexpected adverse events. Follow-up PET scans showed new areas of viability within the infarct scar in CABG patients. Echocardiography measured an average improvement in left ventricular ejection fraction (LVEF) from 25% to 34%. Histological evaluation in four out of five patients who underwent heart transplantation documented survival and engraftment of the skeletal myoblasts within the infarcted myocardium. These interim results demonstrate survival, feasibility, and safety of autologous myoblast transplantation and suggest that this modality may offer a potential therapeutic treatment for end-stage heart disease.     

Human myoblast engraftment is improved in laminin-enriched microenvironment

BACKGROUND: One major challenge in developing cell therapy for muscle diseases is to define the best condition for the recipient's muscle to niche donor cells. We have examined the efficiency of human myoblast transplantation in an immunodeficient animal model, after local irradiation, as well as the potential impact of laminin on myoblast behavior. METHODS: Human myoblasts were injected into preirradiated tibialis anterior muscles from immunodeficient mice. The donor cell engraftment, proliferation, and laminin content within the transplanted muscles were evaluated by immunocytochemistry. Additionally, the effect of laminin upon myoblast proliferation, migration, and survival was ascertained in vitro. RESULTS: Engraftment of human myoblasts into the skeletal muscle of immunodeficient Rag2-/gammac-/C5- mice presubjected to local irradiation provided the best niche for myoblast engraftment, as demonstrated by the number of viable and proliferating donor cells found in the host muscle. Local irradiation significantly enhanced laminin deposition within the recipient's muscle and donor cells were preferentially located in laminin-enriched areas. The same batch of myoblasts used for in vivo injections also responded to laminin in vitro with increased proliferation and cell survival, as well as an improved migratory response. CONCLUSIONS: We show that local irradiation enhances the laminin content in the host muscle microenvironment and provides a better engraftment of human myoblasts. In addition, laminin increases myoblast proliferation, survival, and migration in vitro. These data provide combined in vivo and in vitro evidence that laminin status should be taken into account when designing experimental and clinical cell therapy strategies for muscle disease.     

Primary myogenic cells see the light: improved survival of transplanted myogenic cells following low energy laser irradiation

BACKGROUND AND OBJECTIVES: There is a substantial need for finding new avenues to promote muscle recovery when acute skeletal muscle loss extends beyond the natural capacity of the muscle to recover. Maintenance and regeneration of skeletal muscles depend mainly on resident stem cells known as satellite cells. Nevertheless, there are situations in which a significant loss of muscle tissue exhausts the satellite cell pool. For such cases, cell therapy and tissue engineering are becoming promising alternatives. Thus far, attempts to supplement damaged host muscles with donor satellite cells by means of myoblast transplantation therapy were mostly unsuccessful due to massive and rapid loss of donor cells within few hours after transplantation. This study aims at following the effects of low-energy-laser irradiation on the fate of implanted myoblasts. STUDY DESIGN: Primary myogenic cells, harvested from male rat skeletal muscles, were irradiated with low energy laser, seeded on a biodegradable scaffold and expanded in vitro. The scaffold containing cells was transplanted into partially excised muscles of host female rats. Donor cells were identified in the host muscle tissue, using Y-chromosome in situ hybridization. RESULTS: In this study, we show that laser irradiated donor primary myogenic cells not only survive, but also fuse with host myoblasts to form a host-donor syncytium. CONCLUSIONS: Our data show that the use of low energy laser irradiation (LELI), a non-surgical tool, is a promising means to enhance both the survival and functionality of transplanted primary myogenic cells.     

Induction of ectopic bone formation by using human periosteal cells in combination with a novel scaffold technology

Due to their osteogenic germination potential, periosteum-derived osteoprogenitor cells are a potential source for tissue engineering a bone graft that could be used to regenerate skeletal defects. In this study we evaluated if ectopic bone formation could be induced by a construct made of human periosteal cells and a novel scaffold architecture whose mechanical properties are in the range of cancellous bone. Biopsies from human calvarial periosteum were harvested and cells were isolated from the inner cambial layer. Fifty thousand periosteal cells were seeded into the scaffolds measuring 6 x 6 x 2 mm. The cell-scaffold constructs were cultured for a period of 3 weeks prior to implantation into balb C nude mice. Mice were sacrificed and implants were analyzed 6 and 17 weeks postoperatively. Immunohistochemical analysis confirmed the osteoblastic phenotype of the seeded cells. Formation of focal adhesions and stress fibers could be observed in both scaffold architectures. Three-dimensional cell proliferation was observed after 2 weeks of culturing with centripetal growth pattern inside the pore network. The deposition of calcified extracellular matrix was observed after 3 weeks of culturing. In vivo, endochondral bone formation with osteoid production was detectable via von Kossa and Osteocalcin staining after 6 and 17 weeks. Histology and SEM revealed that the entire scaffold/bone grafts were penetrated by a vascular network. This study showed the potential of bone tissue engineering by using human periosteal cells in combination with a novel scaffold technology.     

Postnatal myocardial augmentation with skeletal myoblast-based fetal tissue engineering

BACKGROUND: Cardiac anomalies constitute the most common birth defects, many of which involve variable myocardial deficiencies. Therapeutic options for structural myocardial repair remain limited in the neonatal population. This study was aimed at determining whether engineered fetal muscle constructs undergo milieu-dependent transdifferentiation after cardiac implantation, thus becoming a potential means to increase/support myocardial mass after birth. METHODS: Myoblasts were isolated from skeletal muscle specimens harvested from fetal lambs, labeled by transduction with a retrovirus-expressing green fluorescent protein, expanded in vitro, and then seeded onto collagen hydrogels. After birth, animals underwent autologous implantation of the engineered constructs (n = 8) onto the myocardium as an onlay patch. Between 4 and 30 weeks postoperatively, implants were harvested for multiple analyses. RESULTS: Fetal and postnatal survival rates were 89% and 100%, respectively. Labeled cells were identified within the implants at all time points by immunohistochemical staining for green fluorescent protein. At 24 and 30 weeks postimplantation, donor cells double-stained for green fluorescent protein and Troponin I, while losing skeletal (type II) myosin expression. CONCLUSIONS: Fetal skeletal myoblasts engraft in native myocardium up to 30 weeks after postnatal, autologous implantation as components of engineered onlay patches. These cells also display evidence of time-dependent transdifferentiation toward a cardiomyocyte-like lineage. Further analysis of fetal skeletal myoblast-based constructs for the repair of congenital myocardial defects is warranted.     

Factors affecting functional outcome after autologous skeletal myoblast transplantation

BACKGROUND: This study assessed the extent to which the initial degree of functional impairment and the number of injected cells may influence the functional improvement provided by autologous skeletal myoblast transplantation into infarcted myocardium. METHODS: One week after left coronary artery ligation, 44 rats received into the infarcted scar, autologous skeletal myoblasts expanded in vitro for 7 days (mean, 3.5 x 10(6), n = 21), or culture medium alone (controls, n = 23). Left ventricular function was assessed by two-dimensional echocardiography. RESULTS: When transplanted hearts were stratified according to their baseline ejection fraction, a significant improvement occurred at 2 months in the less than 25% (from 21.4% to 37%), 25% to 35% (from 29% to 43.8%), and in the 35% to 40% (from 37.2% to 41.7%) groups, compared to controls (p = 0.048, 0.0057, and 0.034, respectively), but not in the more than 40% stratum. A significant linear relationship was found between the improvement in ejection fraction and the number of injected myoblasts, both at 1 and 2 months after transplantation (p < 0.0001). CONCLUSIONS: Autologous myoblast transplantation is functionally effective over a wide range of postinfarct ejection fractions, including in the sickest hearts provided that they are injected with a sufficiently high number of cells.     

Optimization of bone tissue engineering in goats: a peroperative seeding method using cryopreserved cells and localized bone formation in calcium phosphate scaffolds

BACKGROUND: Bone tissue engineering by combining cultured bone marrow stromal cells with a porous scaffold is a promising alternative for the autologous bone graft. Drawbacks of the technique include the delay necessary for cell culture and the complicated logistics. We investigated methods to bypass these drawbacks. Furthermore, we investigated the localization of bone formation inside the scaffold. METHODS: Bone marrow stromal cells from seven goats were culture expanded and cryopreserved. One week before surgery, some of the cells were thawed, cultured, and seeded on porous calcium phosphate scaffolds. The constructs were cultured for another week until implantation. The remaining cryopreserved cells were thawed just before implantation and peroperatively resuspended in plasma before combining with the scaffold. Scaffolds impregnated with fresh bone marrow, devitalized cultured constructs, and empty scaffolds served as controls. All samples were implanted in the back muscles of the goats for 9 weeks. RESULTS: Histologic examination showed minimal (<1%) bone in the empty and devitalized scaffolds, 4.2 +/- 5.1 bone area percent in the bone marrow samples, and significantly more bone in both the cultured and peroperatively seeded constructs (11.7 +/- 2.5 and 14.0 +/- 2.0%). The peripheral 350 microm of the implants contained significantly less bone. CONCLUSION: Peroperative preparation of osteogenic constructs with cryopreserved cells is feasible. These constructs yield substantially more bone than the scaffolds alone or scaffolds impregnated with fresh bone marrow. Bone deposition is much less on the scaffold periphery.     

Innate inflammatory cells are not responsible for early death of donor myoblasts after myoblast transfer therapy

BACKGROUND: Myoblast transfer therapy (MTT) is a cell-based gene therapy representing a potential treatment for Duchenne muscular dystrophy. The rapid disappearance of donor myoblasts from transplanted muscles after MTT is one of the most controversial and significant obstacles facing research in this area. Dystrophin-deficient muscles show constitutively high levels of inflammation, thus necessitating an examination of whether inflammatory cells, specifically natural killer (NK) cells, neutrophils, and macrophages, within dystrophic muscle are responsible for poor graft survival. METHODS: Female mdx mice were treated with RB6-8C5 monoclonal antibody, PK136 monoclonal antibody, or clodronate liposomes to systemically deplete neutrophils, NK cells, and macrophages, respectively. After each depletion regimen, the mice and age-matched controls received 5.0 x 10 male myoblasts injected longitudinally into each tibialis anterior muscle. Donor myoblast survival was assessed by Y-chromosome specific quantitative real-time polymerase chain reaction analysis. RESULTS.: The systemic depletion of host neutrophils and NK cells resulted in a transient improvement in donor myoblast survival at 72 hr and 7 days post-MTT, respectively. Systemic depletion of macrophages had no significant beneficial effect on myoblast survival. Overall, the number of detectable male donor myoblasts was similar at time 0 and 1 hr post-MTT; however, there was significant loss by 24 hr (approximately 50%-70%) followed by a continual decline in donor cell numbers. CONCLUSIONS: Neutrophils and macrophages do not seem to play a major role in the rapid death of donor myoblasts after transplantation into dystrophic muscle. NK cells similarly seem to have no significant effect, contrary to earlier findings reported by our group.