Poly(Lactic-Co-Glycolic Acid) Microparticles for the Delivery of Model Drug Compounds for Applications in Vascular Tissue Engineering.

INTRODUCTION: Localized delivery of angiogenesis-promoting factors such as small molecules, nucleic acids, peptides, and proteins to promote the repair and regeneration of damaged tissues remains a challenge in vascular tissue engineering. Current delivery methods such as direct administration of therapeutics can fail to maintain the necessary sustained release profile and often rely on supraphysiologic doses to achieve the desired therapeutic effect. By implementing a microparticle delivery system, localized delivery can be coupled with sustained and controlled release to mitigate the risks involved with the high dosages currently required from direct therapeutic administration. METHODS: For this purpose, poly(lactic-co-glycolic acid) (PLGA) microparticles were fabricated via anti-solvent microencapsulation and the loading, release, and delivery of model angiogenic molecules, specifically a small molecule, nucleic acid, and protein, were assessed in vitro using microvascular fragments (MVFs). RESULTS: The microencapsulation approach utilized enabled rapid spherical particle formation and encapsulation of model drugs of different sizes, all in one method. The addition of a fibrin scaffold, required for the culture of the MVFs, reduced the initial burst of model drugs observed in release profiles from PLGA alone. Lastly, in vitro studies using MVFs demonstrated that higher concentrations of microparticles led to greater co-localization of the model therapeutic (miRNA) with MVFs, which is vital for targeted delivery methods. It was also found that the biodistribution of miRNA using the delivered microparticle system was enhanced compared to direct administration. CONCLUSION: Overall, PLGA microparticles, formulated and loaded with model therapeutic compounds in one step, resulted in improved biodistribution in a model of the vasculature leading to a future in translational revascularization.

Intravenous Autologous Bone Marrow-derived Mesenchymal Stromal Cells Delay Acute Respiratory Distress Syndrome in Swine.

Rationale: Early post injury mitigation strategies in ARDS are in short supply. Treatments with allogeneic stromal cells are administered after ARDS develops, require specialized expertise and equipment, and to date have shown limited benefit. Objectives: Assess the efficacy of immediate post injury intravenous administration of autologous or allogeneic bone marrow-derived mesenchymal stromal cells (MSCs) for the treatment of acute respiratory distress syndrome (ARDS) due to smoke inhalation and burns. Methods: Yorkshire swine (n = 32, 44.3 ± 0.5 kg) underwent intravenous anesthesia, placement of lines, severe smoke inhalation, and 40% total body surface area flame burns, followed by 72 hours of around-the-clock ICU care. Mechanical ventilation, fluids, pressors, bronchoscopic cast removal, daily lung computed tomography scans, and arterial blood assays were performed. After injury and 24 and 48 hours later, animals were randomized to receive autologous concentrated bone marrow aspirate (n = 10; 3 × 106 white blood cells and a mean of 56.6 × 106 platelets per dose), allogeneic MSCs (n = 10; 6.1 × 106 MSCs per dose) harvested from healthy donor swine, or no treatment in injured control animals (n = 12). Measurements and Main Results: The intravenous administration of MSCs after injury and at 24 and 48 hours delayed the onset of ARDS in swine treated with autologous MSCs (48 ± 10 h) versus control animals (14 ± 2 h) (P = 0.004), reduced ARDS severity at 24 (P < 0.001) and 48 (P = 0.003) hours, and demonstrated visibly diminished consolidation on computed tomography (not significant). Mortality at 72 hours was 1 in 10 (10%) in the autologous group, 5 in 10 (50%) in the allogeneic group, and 6 in 12 (50%) in injured control animals (not significant). Both autologous and allogeneic MSCs suppressed systemic concentrations of TNF-α (tumor necrosis factor-α). Conclusions: The intravenous administration of three doses of freshly processed autologous bone marrow-derived MSCs delays ARDS development and reduces its severity in swine. Bedside retrieval and administration of autologous MSCs in swine is feasible and may be a viable injury mitigation strategy for ARDS.

Ascorbic acid diminishes bone morphogenetic protein 2-induced osteogenic differentiation of muscle precursor cells.

INTRODUCTION: Muscle precursor cells (MPC) are integral to the maintenance of skeletal muscle and have recently been implicated in playing a role in bone repair. The primary objective of this study was to understand better the role of oxidative stress during the osteogenic differentiation of MPCs. METHODS: Muscle precursor cells were treated with various combinations of ascorbic acid (AA), bone morphogenetic protein (BMP)-2, and either a superoxide dismutase analog (4-hydroxy-TEMPO [TEMPOL]) or polyethyleneglycol-conjugated catalase. Muscle precursor cell proliferation and differentiation were determined, and alkaline phosphatase activity was measured as an index of osteogenic differentiation. RESULTS: After treatment with 200 µM AA, superoxide was increased 1.5-fold, whereas AA in combination with 100 ng/ml BMP-2 did not increase alkaline phosphatase (ALP) activity. When cells were treated with TEMPOL in combination with 100 ng/ml BMP-2 and 200 µM AA, ALP activity significantly increased. DISCUSSION: These data suggest that increasing oxidative stress with AA induces sublethal oxidative stress that prevents BMP-2-induced osteogenic differentiation of MPCs. Muscle Nerve 59:501-508, 2019.

Alternatives to autograft evaluated in a rabbit segmental bone defect.

PURPOSE: This study was designed to identify strategies for treating bone defects that can be completed on the day of surgery. METHODS: Forty New Zealand white rabbits with unilateral rabbit radius segmental defects (15 mm) were treated with commercially available scaffolds containing either demineralised bone matrix (DBM) or a collagen/beta-tricalcium phosphate composite (Col:ß-TCP); each scaffold was combined with either bone marrow aspirate (BMA) or concentrated BMA (cBMA). Bone regeneration was assessed through radiographic and histological analyses. RESULTS: The concentration of nucleated cells, colony-forming unit-fibroblasts and platelets were increased and haematocrit concentration decreased in cBMA as compared to BMA (p < 0.05). Radiographic analyses of bone formation and defect bridging demonstrated significantly greater bone regeneration in the defects treated with DBM grafts as compared to Col:ß-TCP grafts. The healing of bones treated with Col:ß-TCP was improved when augmented with cBMA. CONCLUSIONS: Scaffolds containing either DBM or Col:ß-TCP with BMA or cBMA are effective same-day strategies available to clinicians for the treatment of bone defects; the latter scaffold may be more effective if combined with cBMA.

Soluble factors from biofilms of wound pathogens modulate human bone marrow-derived stromal cell differentiation, migration, angiogenesis, and cytokine secretion.

BACKGROUND: Chronic, non-healing wounds are often characterized by the persistence of bacteria within biofilms - aggregations of cells encased within a self-produced polysaccharide matrix. Biofilm bacteria exhibit unique characteristics from planktonic, or culture-grown, bacterial phenotype, including diminished responses to antimicrobial therapy and persistence against host immune responses. Mesenchymal stromal cells (MSCs) are host cells characterized by their multifunctional ability to undergo differentiation into multiple cell types and modulation of host-immune responses by secreting factors that promote wound healing. While these characteristics make MSCs an attractive therapeutic for wounds, these pro-healing activities may be differentially influenced in the context of an infection (i.e., biofilm related infections) within chronic wounds. Herein, we evaluated the effect of soluble factors derived from biofilms of clinical isolates of Staphylococcus aureus and Pseudomonas aeruginosa on the viability, differentiation, and paracrine activity of human MSCs to evaluate the influence of biofilms on MSC activity in vitro. RESULTS: Exposure of MSCs to biofilm-conditioned medias of S. aureus and P. aeruginosa resulted in reductions in cell viability, in part due to activation of apoptosis. Similarly, exposure to soluble factors from biofilms was also observed to diminish the migration ability of cells and to hinder multi-lineage differentiation of MSCs. In contrast to these findings, exposure of MSCs to soluble factors from biofilms resulted in significant increases in the release of paracrine factors involved in inflammation and wound healing. CONCLUSIONS: Collectively, these findings demonstrate that factors produced by biofilms can negatively impact the intrinsic properties of MSCs, in particular limiting the migratory and differentiation capacity of MSCs. Consequently, these studies suggest use/application of stem-cell therapies in the context of infection may have a limited therapeutic effect.

Transplantation of devitalized muscle scaffolds is insufficient for appreciable de novo muscle fiber regeneration after volumetric muscle loss injury.

Volumetric muscle loss (VML) is a traumatic and functionally debilitating muscle injury with limited treatment options. Developmental regenerative therapies for the repair of VML typically comprise an ECM scaffold. In this study, we tested if the complete reliance on host cell migration to a devitalized muscle scaffold without myogenic cells is sufficient for de novo muscle fiber regeneration. Devitalized (muscle ECM with no living cells) and, as a positive control, vital minced muscle grafts were transplanted to a VML defect in the tibialis anterior muscle of Lewis rats. Eight weeks post-injury, devitalized grafts did not appreciably promote de novo muscle fiber regeneration within the defect area, and instead remodeled into a fibrotic tissue mass. In contrast, transplantation of vital minced muscle grafts promoted de novo muscle fiber regeneration. Notably, pax7+ cells were absent in remote regions of the defect site repaired with devitalized scaffolds. At 2 weeks post-injury, the devitalized grafts were unable to promote an anti-inflammatory phenotype, while vital grafts appeared to progress to a pro-regenerative inflammatory response. The putative macrophage phenotypes observed in vivo were supported in vitro, in which soluble factors released from vital grafts promoted an M2-like macrophage polarization, whereas devitalized grafts failed to do so. These observations indicate that although the remaining muscle mass serves as a source of myogenic cells in close proximity to the defect site, a devitalized scaffold without myogenic cells is inadequate to appreciably promote de novo muscle fiber regeneration throughout the VML defect.

Accelerated functional recovery after skeletal muscle ischemia-reperfusion injury using freshly isolated bone marrow cells.

BACKGROUND: Relatively little information exists regarding the usefulness of bone marrow-derived cells for skeletal muscle ischemia-reperfusion injury (I/R), especially when compared with I/R that occurs in other tissues. The objectives of this study were to evaluate the ability of freshly isolated bone marrow cells to home to injured skeletal muscle and to determine their effects on muscle regeneration. MATERIALS AND METHODS: Freshly isolated lineage-depleted bone marrow cells (Lin(-) BMCs) were injected intravenously 2 d after I/R. Bioluminescent imaging was used to evaluate cell localization for up to 28 d after injury. Muscle function, the percentage of fibers with centrally located nuclei, and the capillary-to-fiber ratio were evaluated 14 d after delivery of either saline (Saline) or saline containing Lin(-) BMCs (Lin(-) BMCs). RESULTS: Bioluminescence was higher in the injured leg than the contralateral control leg for up to 7 d after injection (P < 0.05) suggestive of cell homing to the injured skeletal muscle. Fourteen days after injury, there was a significant improvement in maximal tetanic torque (40% versus 22% deficit; P < 0.05), a faster rate of force production (+dP/dt) (123.6 versus 94.5 Nmm/S; P < 0.05), and a reduction in the percentage of fibers containing centrally located nuclei (40 versus 17%; P < 0.05), but no change in the capillary-to-fiber ratio in the Lin(-) BMC as compared with the Saline group. CONCLUSIONS: The homing of freshly isolated BMCs to injured skeletal muscle after I/R is associated with an increase in functional outcomes.

Characterization and multilineage potential of cells derived from isolated microvascular fragments.

BACKGROUND: A number of therapies are being developed that use microvessels isolated from adipose tissue (microvascular fragments [MVFs]) to improve tissue perfusion and implant survival. Because it has been demonstrated that stem cells are associated with microvessels, the purpose of these studies was to gain further insight into the stem cells associated with MVFs to better understand their therapeutic potential. MATERIALS AND METHODS: Cells derived from MVF explants were compared with adipose-derived stem cells (ASCs) based on the expression of cell surface proteins for mesenchymal stem cells and their capacity for angiogenic, neurogenic, adipogenic, and osteogenic differentiation. RESULTS: The expression of cell surface proteins for mesenchymal stem cell markers was similar between MVF-derived cells and ASCs; however, the increase in markers consistent with endothelial cells and pericytes was accompanied by an improved ability to form capillary-like networks when cultured on matrigel. MVF-derived cells had increased neuregulin, leptin, and osteopontin expression compared with ASCs when exposed to neurogenic, adipogenic, and osteogenic induction media, respectively. CONCLUSIONS: The stem cell functionality of cells derived from MVFs is retained after their isolation. This helps to explain the ability of MVFs to improve tissue perfusion and has implications for the use of MVFs as a means to deliver stem cells within their niche.

Skeletal muscle satellite cell migration to injured tissue measured with 111In-oxine and high-resolution SPECT imaging.

The delivery of adult skeletal muscle stem cells, called satellite cells, to several injured muscles via the circulation would be useful, however, an improved understanding of cell fate and biodistribution following their delivery is important for this goal to be achieved. The objective of this study was to evaluate the ability of systemically delivered satellite cells to home to injured skeletal muscle using single-photon emission computed tomography (SPECT) imaging of (111)In-labeled satellite cells. Satellite cells labeled with (111)In-oxine and green fluorescent protein (GFP) were injected intravenously after bupivicaine-induced injury to the tibialis anterior muscle. Animals were imaged with a high-resolution SPECT system called FastSPECT II for up to 7 days after transplantation. In vivo FastSPECT II imaging demonstrated a three to five-fold greater number of transplanted satellite cells in bupivicaine-injured muscle as compared to un-injured muscle after transplantation; a finding that was verified through autoradiograph analysis and quantification of GFP expression. Satellite cells also accumulated in other organs including the lung, liver, and spleen, as determined by biodistribution measurements. These data support the ability of satellite cells to home to injured muscle and support the use of SPECT and autoradiograph imaging techniques to track systemically transplanted (111)In labeled satellite cells in vivo, and suggest their homing may be improved by reducing their entrapment in filter organs.

Satellite cell functional alterations following cutaneous burn in rats include an increase in their osteogenic potential.

BACKGROUND: Significant consequences of severe burn include skeletal muscle atrophy and heterotopic ossification (HO). The cellular mechanisms underlying either of these conditions are not known. Whether the functionality of satellite cells stem cells resident in skeletal muscle is affected by changes in circulatory factors following burn was determined to better understand their role in atrophy and HO. MATERIALS AND METHODS: Serum (20%) from sham-treated animals or burned animals (40% total body surface area full-thickness burn) was used to culture satellite cells isolated from either sham or burn animals. Satellite cells were separated based on fiber type (i.e., fast-twitch or slow-twitch in some cases). To gain greater insight into the potential role for satellite cells in controlling muscle mass following burn, the effect of serum taken from burn animals on satellite cell proliferation, migration, and myogenic differentiation was evaluated. Osteogenic differentiation was assessed to evaluate the potential of satellite cells to contribute to HO. RESULTS: Burn serum (BS) increased the proliferative capacity of cells from fast-twitch muscle, and the migratory capacity of satellite cells taken from both fast- and slow-twitch muscles. BS increased both the myogenic and osteogenic differentiation of satellite cells taken from both sham and burn animals. CONCLUSIONS: The unexpected increase in myogenic functionality of satellite cells with BS is difficult to rectify, given the degree of atrophy that occurs. However, the increased osteogenic capacity of satellite cells with BS suggests they may play a role in burn-induced HO.

Staphylococcus aureus biofilms decrease osteoblast viability, inhibits osteogenic differentiation, and increases bone resorption in vitro.

BACKGROUND: Osteomyelitis is a severe and often debilitating disease characterized by inflammatory destruction of bone. Despite treatment, chronic infection often develops which is associated with increased rates of treatment failure, delayed osseous-union, and extremity amputation. Within affected bone, bacteria exist as biofilms, however the impact of biofilms on osteoblasts during disease are unknown. Herein, we evaluated the effect of S. aureus biofilms on osteoblast viability, osteogenic potential, and the expression of the pro-osteoclast factor, receptor activator of NF-kB ligand (RANK-L). METHODS: Osteoblasts were exposed to biofilm conditioned media (BCM) from clinical wound isolates of Staphylococcus aureus under normal growth and osteogenic conditions to assess cellular viability and osteoblast differentiation, respectively. Cell viability was evaluated using a live/dead assay and by quantifying total cellular DNA at days 0, 1, 3, 5, and 7. Apoptosis following treatment with BCM was measured by flow-cytometry using the annexin V-FITC/PI apoptosis kit. Osteogenic differentiation was assessed by measuring alkaline phosphatase activity and intracellular accumulation of calcium and osteocalcin for up to 21 days following exposure to BCM. Expression of genes involved in osteogenic differentiation and osteoclast regulation, were also evaluated by quantitative real-time PCR. RESULTS: BCM from clinical strains of S. aureus reduced osteoblast viability which was accompanied by an increase in apoptosis. Osteogenic differentiation was significantly inhibited following treatment with BCM as indicated by decreased alkaline phosphatase activity, decreased intracellular accumulation of calcium and inorganic phosphate, as well as reduced expression of transcription factors and genes involved in bone mineralization in viable cells. Importantly, exposure of osteoblasts to BCM resulted in up-regulated expression of RANK-L and increase in the RANK-L/OPG ratio compared to the untreated controls. CONCLUSIONS: Together these studies suggest that soluble factors produced by S. aureus biofilms may contribute to bone loss during chronic osteomyelitis simultaneously by: (1) reducing osteoblast viability and osteogenic potential thereby limiting new bone growth and (2) promoting bone resorption through increased expression of RANK-L by osteoblasts. To our knowledge these are the first studies to demonstrate the impact of staphylococcal biofilms on osteoblast function, and provide an enhanced understanding of the pathogenic role of staphylococcal biofilms during osteomyelitis.

Diabetes diminishes muscle precursor cell-mediated microvascular angiogenesis.

The skeletal muscles of Type II diabetic (T2D) patients can be characterized by a reduced vessel density, corresponding to deficiencies in microvascular angiogenesis. Interestingly, T2D also inhibits the function of many myogenic cells resident within skeletal muscle, including satellite cells, which are well-known for the role they play in maintaining homeostasis. The current study was undertaken to gain a better understanding of the mechanisms whereby satellite cell progeny, muscle precursor cells (MPCs), influence microvascular angiogenesis. Network growth and the expression of genes associated with angiogenesis were reduced when microvessels were treated with conditioned media generated by proliferating MPCs isolated from diabetic, as compared to control rat skeletal muscle, a phenomenon that was also observed when myoblasts from control or diabetic human skeletal muscle were used. When only exosomes derived from diabetic or control MPCs were used to treat microvessels, no differences in microvascular growth were observed. An evaluation of the angiogenesis factors in control and diabetic MPCs revealed differences in Leptin, vascular endothelial growth factor (VEGF), IL1-ß, interleukin 10, and IP-10, and an evaluation of the MPC secretome revealed differences in interleukin 6, MCP-1, VEGF, and interleukin 4 exist. Angiogenesis was also reduced in tissue-engineered skeletal muscles (TE-SkM) containing microvessels when they were generated from MPCs isolated from diabetic as compared to control skeletal muscle. Lastly, the secretome of injured control, but not diabetic, TE-SkM was able to increase VEGF and increase microvascular angiogenesis. This comprehensive analysis of the interaction between MPCs and microvessels in the context of diabetes points to an area for alleviating the deleterious effects of diabetes on skeletal muscle.

Three-Dimensional Culture of Vascularized Thermogenic Adipose Tissue from Microvascular Fragments.

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for the design of personalized microtissues for health screening and drug testing. Current strategies are often quite complex and fail to accurately fully depict the multicellular and functional properties of thermogenic adipose tissue. Microvascular fragments, small intact microvessels comprised of arteriole, venules, and capillaries isolated from adipose tissue, serve as a single autologous source of cells that enable vascularization and adipose tissue formation. This article describes methods for optimizing culture conditions to enable the generation of three-dimensional, vascularized, and functional thermogenic adipose tissues from microvascular fragments, including protocols for isolating microvascular fragments from adipose tissue and culture conditions. Additionally, best practices are discussed, as are techniques for characterizing the engineered tissues, and sample results from both rodent and human microvascular fragments are provided. This approach has the potential to be utilized for the understanding and development of treatments for obesity and metabolic disease.

Slow-adhering stem cells derived from injured skeletal muscle have improved regenerative capacity.

A wide variety of myogenic cell sources have been used for repair of injured and diseased muscle including muscle stem cells, which can be isolated from skeletal muscle as a group of slow-adhering cells on a collagen-coated surface. The therapeutic use of muscle stem cells for improving muscle regeneration is promising; however, the effect of injury on their characteristics and engraftment potential has yet to be described. In the present study, slow-adhering stem cells (SASCs) from both laceration-injured and control noninjured skeletal muscles in mice were isolated and studied. Migration and proliferation rates, multidifferentiation potentials, and differences in gene expression in both groups of cells were compared in vitro. Results demonstrated that a larger population of SASCs could be isolated from injured muscle than from control noninjured muscle. In addition, SASCs derived from injured muscle demonstrated improved migration, a higher rate of proliferation and multidifferentiation, and increased expression of Notch1, STAT3, Msx1, and MMP2. Moreover, when transplanted into dystrophic muscle in MDX/SCID mice, SASCs from injured muscle generated greater engraftments with a higher capillary density than did SASCs from control noninjured muscle. These data suggest that traumatic injury may modify stem cell characteristics through trophic factors and improve the transplantation potential of SASCs in alleviating skeletal muscle injuries and diseases.

Engineering Human Beige Adipose Tissue.

In this study, we described a method for generating functional, beige (thermogenic) adipose microtissues from human microvascular fragments (MVFs). The MVFs were isolated from adipose tissue acquired from adults over 50 years of age. The tissues express thermogenic gene markers and reproduce functions essential for the potential therapeutic impact of beige adipose tissues such as enhanced lipid metabolism and increased mitochondrial respiration. MVFs serve as a potential single, autologous source of cells that can be isolated from adult patients, induced to recreate functional aspects of beige adipose tissue and enable rapid vascularization post-transplantation. This approach has the potential to be used as an autologous therapy for metabolic diseases or as a model for the development of a personalized approach to high-throughput drug development/screening for adipose tissue.

Adipogenic Differentiation Alters Properties of Vascularized Tissue-Engineered Skeletal Muscle.

Advances in the engineering of comprehensive skeletal muscle models in vitro will improve drug screening platforms and can lead to better therapeutic approaches for the treatment of skeletal muscle injuries. To this end, a vascularized tissue-engineered skeletal muscle (TE-SkM) model that includes adipocytes was developed to better emulate the intramuscular adipose tissue that is observed in skeletal muscles of patients with diseases such as diabetes. Muscle precursor cells cultured with and without microvessels derived from adipose tissue (microvascular fragments) were used to generate TE-SkM constructs, with and without a microvasculature, respectively. TE-SkM constructs were treated with adipogenic induction media to induce varying levels of adipogenesis. With a delayed addition of induction media to allow for angiogenesis, a robust microvasculature in conjunction with an increased content of adipocytes was achieved. The augmentation of vascularized TE-SkM constructs with adipocytes caused a reduction in maturation (compaction), mechanical integrity (Young's modulus), and myotube and vessel alignment. An increase in basal glucose uptake was observed in both levels of adipogenic induction, and a diminished insulin-stimulated glucose uptake was associated with the higher level of adipogenic differentiation and the greater number of adipocytes.

Engineering Functional Vascularized Beige Adipose Tissue from Microvascular Fragments of Models of Healthy and Type II Diabetes Conditions.

Engineered beige adipose tissues could be used for screening therapeutic strategies or as a direct treatment for obesity and metabolic disease. Microvascular fragments are vessel structures that can be directly isolated from adipose tissue and may contain cells capable of differentiation into thermogenic, or beige, adipocytes. In this study, culture conditions were investigated to engineer three-dimensional, vascularized functional beige adipose tissue using microvascular fragments isolated from both healthy animals and a model of type II diabetes (T2D). Vascularized beige adipose tissues were engineered and exhibited increased expression of beige adipose markers, enhanced function, and improved cellular respiration. While microvascular fragments isolated from both lean and diabetic models were able to generate functional tissues, differences were observed in regard to vessel assembly and tissue function. This study introduces an approach that could be employed to engineer vascularized beige adipose tissues from a single, potentially autologous source of cells.

Effects of transforming growth factor-beta (TGF-ß1) on satellite cell activation and survival during oxidative stress.

The regulation of adult skeletal muscle repair and regeneration is largely due to the contribution of resident adult myogenic precursor cells called satellite cells. The events preceding their participation in muscle repair include activation (exit from quiescence), proliferation, and differentiation. This study examined the effects of transforming growth factor-beta (TGF-ß1) on satellite cell activation, determined whether TGF-ß1 could maintain quiescence in the presence of hepatocyte growth factor (HGF), and whether the regulation of satellite cell activation with TGF-ß1 improves the ability of satellite cells to withstand oxidative stress. The addition of TGF-ß1 during early satellite cell activation (0-48 h) or during the proliferative phase (48-96 h) maintained and induced satellite cell quiescence, respectively, as determined by myogenic differentiation (MyoD) protein expression. TGF-ß1 also attenuated satellite cell activation when used with HGF. Finally, the role of quiescence in protecting cells against oxidative stress was examined. TGF-ß1 treatment and the low pH satellite cell preparation procedure, a technique that forestalls spontaneous activation in vitro, both enhanced survival of cultured satellite cells following hydrogen peroxide treatment. These findings indicate that TGF-ß1 is capable of maintaining and inducing satellite cell quiescence and suggest methods to maintain satellite cell quiescence may improve their transplantation efficiency.

Treatment of tourniquet-induced ischemia reperfusion injury with muscle progenitor cells.

BACKGROUND: Acute ischemia reperfusion injury (IRI) results in muscle atrophy and functional loss. Although studies have shown that stem cells can improve muscle function in chronic ischemia caused by vascular diseases, none investigated whether stem cells can improve muscle function following acute IRI. The primary purpose of this study was to determine whether transplantation of muscle progenitor cells (MPCs) improves recovery of muscle function after tourniquet (TK) induced IRI. METHODS: IRI was induced in rat hind limb muscles with a pneumatic TK (250 mmHg) for 3 h. Rats were then divided into two groups; receiving either intramuscular injection of MPCs or vehicle control into the injured tibialis anterior muscle 48 h after tourniquet application. Muscle mass, isometric contractile properties, and selected histologic properties were evaluated at 2 wk after ischemia. RESULTS: IRI resulted in significant reductions in absolute muscle force (N) and specific muscle force (N/cm(2)). MPC treatment significantly prevented the loss in muscle specific force compared with vehicle controls. The mass and cross sectional areas of the muscles were similar between treatment groups. Histologic results showed that a small number of transplanted cells differentiated and formed muscle fibers, which could potentially contribute to force generation. IRI caused significant fibrosis and inflammation, both of which could affect muscle-specific force, of which inflammation was reduced by MPCs treatment. CONCLUSIONS: Intramuscular injection of MPCs may provide a beneficial treatment for improving functional recovery following IRI, and the beneficial effects are mainly through improving muscle quality (specific force) but not quantity (mass).

Diabetic Conditions Confer Metabolic and Structural Modifications to Tissue-Engineered Skeletal Muscle.

Skeletal muscle is a tissue that is directly involved in the progression and persistence of type 2 diabetes (T2D), a disease that is becoming increasingly common. Gaining better insight into the mechanisms that are affecting skeletal muscle dysfunction in the context of T2D has the potential to lead to novel treatments for a large number of patients. Through its ability to emulate skeletal muscle architecture while also incorporating aspects of disease, tissue-engineered skeletal muscle (TE-SkM) has the potential to provide a means for rapid high-throughput discovery of therapies to treat skeletal muscle dysfunction, to include that which occurs with T2D. Muscle precursor cells isolated from lean or obese male Zucker diabetic fatty rats were used to generate TE-SkM constructs. Some constructs were treated with adipogenic induction media to accentuate the presence of adipocytes that is a characteristic feature of T2D skeletal muscle. The maturity (compaction and creatine kinase activity), mechanical integrity (Young's modulus), organization (myotube orientation), and metabolic capacity (insulin-stimulated glucose uptake) were all reduced by diabetes. Treating constructs with adipogenic induction media increased the quantity of lipid within the diabetic TE-SkM constructs, and caused changes in construct compaction, cell orientation, and insulin-stimulated glucose uptake in both lean and diabetic samples. Collectively, the findings herein suggest that the recapitulation of structural and metabolic aspects of T2D can be accomplished by engineering skeletal muscle in vitro.