Poly(lactic-co-glycolic acid) Microparticles for the Delivery of Model Drug Compounds for Applications in Vascular Tissue Engineering.

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 controlled release to mitigate the risks involved with the high dosages currently required from direct therapeutic administration. For this purpose, poly(lactic-co-glycolic acid) microparticles were fabricated via antisolvent 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 (MVF). 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. Overall, poly(lactic-co-glycolic acid) 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.

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.

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.

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.

Extracellular vesicles derived from cardiosphere-derived cells as a potential antishock therapeutic.

BACKGROUND: Extracellular vesicles (EVs) isolated from cardiosphere-derived cells (CDC-EVs) are coming to light as a unique cell-free therapeutic. Because of their novelty, however, there still exist prominent gaps in knowledge regarding their therapeutic potential. Herein the therapeutic potential of CDC-EVs in a rat model of acute traumatic coagulopathy induced by multiple injuries and hemorrhagic shock is outlined. METHODS: Extracellular vesicle surface expression of procoagulant molecules (tissue factor and phosphatidylserine) was evaluated by flow cytometry. Extracellular vesicle thrombogenicity was tested using calibrated thrombogram, and clotting parameters were assessed using a flow-based adhesion model simulating blood flow over a collagen-expressing surface. The therapeutic efficacy of EVs was then determined in a rat model of acute traumatic coagulopathy induced by multiple injuries and hemorrhagic shock. RESULTS: Extracellular vesicles isolated from cardiosphere-derived cells are not functionally procoagulant and do not interfere with platelet function. In a rat model of multiple injuries and hemorrhagic shock, early administration of EVs significantly reduced the elevation of lactate and creatinine and did not significantly enhance coagulopathy in rats with acute traumatic coagulopathy. CONCLUSION: The results of this study are of great relevance to the development of EV products for use in combat casualty care, as our studies show that CDC-EVs have the potential to be an antishock therapeutic if administered early. These results demonstrate that research using CDC-EVs in trauma care needs to be considered and expanded beyond their reported cardioprotective benefits.

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.

Scaffold Architecture and Matrix Strain Modulate Mesenchymal Cell and Microvascular Growth and Development in a Time Dependent Manner.

BACKGROUND: Volumetric tissue-engineered constructs are limited in development due to the dependence on well-formed vascular networks. Scaffold pore size and the mechanical properties of the matrix dictates cell attachment, proliferation and successive tissue morphogenesis. We hypothesize scaffold pore architecture also controls stromal-vessel interactions during morphogenesis. METHODS: The interaction between mesenchymal stem cells (MSCs) seeded on hydroxyapatite scaffolds of 450, 340, and 250 µm pores and microvascular fragments (MVFs) seeded within 20 mg/mL fibrin hydrogels that were cast into the cell-seeded scaffolds, was assessed in vitro over 21 days and compared to the fibrin hydrogels without scaffold but containing both MSCs and MVFs. mRNA sequencing was performed across all groups and a computational mechanics model was developed to validate architecture effects on predicting vascularization driven by stiffer matrix behavior at scaffold surfaces compared to the pore interior. RESULTS: Lectin staining of decalcified scaffolds showed continued vessel growth, branching and network formation at 14 days. The fibrin gel provides no resistance to spread-out capillary networks formation, with greater vessel loops within the 450 µm pores and vessels bridging across 250 µm pores. Vessel growth in the scaffolds was observed to be stimulated by hypoxia and successive angiogenic signaling. Fibrin gels showed linear fold increase in VEGF expression and no change in BMP2. Within scaffolds, there was multiple fold increase in VEGF between days 7 and 14 and early multiple fold increases in BMP2 between days 3 and 7, relative to fibrin. There was evidence of yap/taz based hippo signaling and mechanotransduction in the scaffold groups. The vessel growth models determined by computational modeling matched the trends observed experimentally. CONCLUSION: The differing nature of hypoxia signaling between scaffold systems and mechano-transduction sensing matrix mechanics were primarily responsible for differences in osteogenic cell and microvessel growth. The computational model implicated scaffold architecture in dictating branching morphology and strain in the hydrogel within pores in dictating vessel lengths.

Divergent effects of myogenic differentiation and diabetes on the capacity for muscle precursor cell adipogenic differentiation in a fibrin matrix.

The development of ectopic adipose tissue in skeletal muscle is associated with several skeletal muscle and metabolic pathologies, including Type II Diabetes Mellitus. The adipogenic differentiation of muscle precursor cells (MPCs) has been postulated to occur in skeletal muscle in vivo in a three-dimensional (3-D) configuration; therefore, it is appropriate to investigate this phenomenon using 3-D matrices in vitro. The capacity for MPC adipogenic differentiation in a 3-D environment was investigated in fibrin hydrogels by treating MPCs derived from healthy or diabetic animals with adipogenic induction medias that differed in their ability to increase lipid accumulation and activate the expression of genes associated with adipogenic differentiation (peroxisome proliferator-activated receptor gamma (PPARG), adiponectin (ADIPOQ), and fatty acid synthase (FAS)). The capacity for adipogenic differentiation was diminished, but not prevented, if myogenic differentiation preceded MPC exposure to adipogenic induction conditions. Conversely, adipogenic differentiation was greater in hydrogels containing MPCs from diabetic rats as compared to those derived from lean rats, as evidenced by an increase in lipid accumulation and adipogenic gene expression. Collectively, the data herein support a role for the MPCs in adipogenesis in a 3-D environment and that they may contribute to the ectopic accumulation of adipose tissue. The observation that the potential for adipogenic differentiation is maintained even after a period of myogenic differentiation alludes to the possibility that adipogenesis may occur during different phases of muscle development. Finally, the increase in adipogenic differentiation in hydrogels containing MPCs derived from diabetic animals provides strong evidence that a pathological environment in vivo increases their capacity for adipogenesis.

Injections of concentrated bone marrow aspirate as treatment for Discogenic pain: a retrospective analysis.

BACKGROUND: There are an overwhelming number of patients suffering from low back pain (LBP) resulting from disc pathology. Although several strategies are being developed pre-clinically, simple strategies to treat the large number of patients currently affected is still needed. One option is to use concentrated bone marrow aspirate (cBMA), which may be effective due to its intrinsic stem cells and growth factors. METHODS: Thirty-three patients who received intradiscal injections of cBMA to relieve LBP were followed up based on Numeric Rating Scale (NRS), Oswestry Low Back Pain Disability Index (ODI), and Short Form-36 Health Survey (SF-36) scores. Patients were also subdivided into those with a pre-injection NRS > 5 and pre-injection NRS ≤ 5. The proportion of patients demonstrating at least 50% improvement (and 95% confidence intervals) from baseline at five follow-up visits for each outcome was evaluated. RESULTS: At least 50% improvement in NRS was observed for 13.8, 45.8, 41.1, 23.5, and 38.9% of patients across five follow-up visits, out to 1 year. When stratified by high (> 5) versus low (≤ 5) baseline NRS scores, the values were 14.3, 45.5, 71.4, 22.2, and 44.4% among those with high baseline pain, and 13.3, 46.2, 20.0, 25.0, and 33.3% among those with low baseline pain. The 50% improvement rates across visits were 4.3, 28.6, 30.0, 22.2, and 30.8% for SF-36, and 4.2, 26.7, 36.4, 55.6, and 30.8% for ODI. CONCLUSIONS: Intradiscal cBMA injections may be effective to reduce pain and improve function. Patients with relatively higher initial pain may have potential for greatest improvement.

Human mesenchymal stromal cell source and culture conditions influence extracellular vesicle angiogenic and metabolic effects on human endothelial cells in vitro.

BACKGROUND: Mesenchymal stem/stromal cell (MSC)-derived extracellular vesicles (EVs) are a possible cell-free alternative to MSCs because they retain the regenerative potential of MSCs, while still mitigating some of their limitations (such as the possible elicitation of host immune responses). The promotion and restoration of angiogenesis, however, is an important component in treating trauma-related injuries, and has not been fully explored with EVs. Herein, we describe the effects of monolayer adipose-derived EVs, spheroid adipose-derived EVs (SAd-EVs), monolayer bone marrow-derived EVs (MBM-EVs), and spheroid bone marrow-derived EVs (SBM-EVs) on human umbilical vein endothelial cell (HUVEC) tube formation and mitochondrial respiration. METHODS: The successful isolation of EVs derived from adipose MSCs or bone marrow MSCs in monolayer or spheroid cultures was confirmed by NanoSight (particle size distribution) and Western blot (surface marker expression). The EV angiogenic potential was measured using a 24-hour HUVEC tube formation assay. The EV effects on HUVEC mitochondrial function were evaluated using the Seahorse respirometer machine. RESULTS: The number of junctions, branches, and the average length of branches formed at 24 hours of tube formation were significantly affected by cell and culture type; overall adipose-derived EVs outperformed bone marrow-derived EVs, and spheroid-derived EVs outperformed monolayer-derived EVs. Additionally, adipose-derived EVs resulted in significantly increased HUVEC mitochondrial maximal respiration and adenosine triphosphate (ATP) production, while only MBM-EVs negatively impacted HUVEC proton leak. CONCLUSION: Adipose-derived EVs promoted HUVEC tube formation significantly more than bone marrow-derived EVs, while also maximizing HUVEC mitochondria function. Results demonstrate that, as with MSC therapies, it is possible to tailor EV culture and production to optimize therapeutic potential. LEVEL OF EVIDENCE: Basic or Foundational Research.

A Straightforward Approach to Engineer Vascularized Adipose Tissue Using Microvascular Fragments.

There is a need to overcome the donor-site morbidity and loss of volume over time that accompanies the current clinical approaches to treat soft tissue defects caused by disease and trauma. The development of bioactive constructs that can regenerate adipose tissue have made great progress toward addressing the limitations of current therapies, but their lack of vascularization and ability to meet the significant dimension requirements of tissue defects limit their clinical translatability. Microvascular fragments (MVFs) can form extensive vascular networks and contain resident cells that have the ability to differentiate into adipocytes. Therefore, the objective of this study was to determine if vascularized adipose tissue could be engineered using a fibrin-based hydrogel containing MVFs as the sole source of microvessels and adipocyte-forming cells. The potential for MVFs from different fat depots (epididymal, inguinal, and subcutaneous) to form microvascular networks and generate adipocytes when exposed to growth media (GM), adipogenic differentiation media (ADM), or when treated with GM before adipogenic induction (i.e., they were allowed to presprout before adipogenic induction) was evaluated. MVFs treated with adipogenic induction media, both with and without presprouting, contained lipid droplets, had an increase in expression levels of genes associated with adipogenesis (adiponectin and fatty acid synthase [FAS]), and had an increased rate of lipolysis. MVFs allowed to presprout before ADM treatment maintained their ability to form vascular networks while maintaining an elevated lipid content, adipogenic gene expression, and lipolysis rate. Collectively, these results support the contention that MVFs can serve as the sole source of biologic material for creating a vascularized adipose tissue scaffold. Impact statement Microvascular fragments have both the ability to form extensive vascular networks and function as a source of adipocytes. These phenomena were exploited as vascularized adipose tissue was generated by first allowing for a period of angiogenesis before the adipogenic induction. This strategy has the ability to provide a means of both improving soft tissue reconstruction while also serving as a model to better understand adipose tissue expansion.

The effects of cell type and culture condition on the procoagulant activity of human mesenchymal stromal cell-derived extracellular vesicles.

BACKGROUND: Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) have great potential as a cell-free therapy in wound healing applications. Because EV populations are not equivalent, rigorous characterization is needed before clinical use. Although there has been much focus on their RNA composition and regenerative capabilities, relatively less is known regarding the effects of MSC cell type (adipose tissue [Ad-MSCs] or bone marrow [BM-MSCs]) and culture condition (monolayer or spheroid) on MSC-EV performance, including characteristics related to their ability to promote coagulation, which could determine EV safety if administered intravenously. METHODS: The successful isolation of EVs derived from Ad-MSCs or BM-MSCs cultured in either monolayer or spheroid cultures was confirmed by NanoSight (particle size distribution) and Western blot (surface marker expression). Extracellular vesicle surface expression of procoagulant molecules (tissue factor and phosphatidylserine) was evaluated by flow cytometry. Extracellular vesicle thrombogenicity was tested using calibrated thrombogram, and clotting parameters were assessed using thromboelastography and a flow-based adhesion model simulating blood flow over a collagen-expressing surface. RESULTS: The MSC cell type and culture condition did not impact EV size distribution. Extracellular vesicles from all groups expressed phosphatidylserine and tissue factor on their surfaces were functionally thrombogenic and tended to increase clotting rates compared to the negative control of serum-free media without EVs. On average, EVs did not form significantly larger or stronger clots than the negative control, regardless of cell source or culture condition. Additionally, EVs interfered with platelet adhesion in an in vitro flow-based assay. CONCLUSION: Adipose-derived EVs were more thrombogenic and expressed higher amounts of phosphatidylserine. Our findings suggest that, like intact MSCs, source variability among EVs is an important factor when considering EVs for potential therapeutic purposes. LEVEL OF EVIDENCE: Therapeutic care management, level II.

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.

Natural polymeric hydrogel evaluation for skeletal muscle tissue engineering.

Although skeletal muscle has a remarkable ability to repair/regenerate after most types of injuries, there is limited regeneration after volumetric muscle loss (VML). A number of scaffold materials have been used in the development of grafts to treat VML, however, there is still a need to better understand the most appropriate material with regards to its ability to maintain mechanical integrity while also supporting myogenesis. Five commonly used natural polymeric materials (Collagen I, Agarose, Alginate, Fibrin, and Collagen Chitosan) used in skeletal muscle tissue engineering grafts were evaluated for their mechanical properties and myogenic capacity. Rheological properties, water absorption rates, degradation stability, tensile characteristics, and the ability to support in vitro myogenesis were compared in all five materials. Collagen, Collagen Chitosan, and Fibrin demonstrated high elasticity and 100% stretch without failure, Agarose was the most brittle (20% max stretch), and Alginate demonstrated poor handleabilty. While Collagen was supportive of myogenesis, overall, Fibrin demonstrated the highest myogenic potential as indicated by the earliest and highest increases in myogenin and myosin heavy chain mRNA in satellite cells along with the most extensive myotube development as evaluated with immunohistochemistry. The findings herein support the notion that under the conditions used in this study, Fibrin is the most suitable scaffold for the development of scaffolds for skeletal muscle tissue engineering. Future studies are required to determine whether the differences in mechanical properties and myogenic potential observed in vitro in the current study translate to better skeletal muscle development in a VML injury model. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 672-679, 2018.

A Retrospective Look at Integrating a Novel Regenerative Medicine Approach in Plastic Limb Reconstruction.

Full-thickness wounds that have rendered patients candidates for amputation may require techniques that may include a combinatorial approach above traditional standard of care. The purpose of this retrospective study was to evaluate the effectiveness of an innovative approach whereby several therapies were combined to avoid amputation. Patients with full-thickness wounds who were previously recommended for amputation and were treated with the combinatorial approach of muscle flap reconstruction and concentrated bone marrow aspirate, platelet-rich plasma, INTEGRA Wound matrix, vacuum-assisted closure, and split-thickness skin grafts were assessed retrospectively. The mean age of the patients identified was 48 years (range, 34-66 years). The average size of the defects was 19.6 cm2. All defects were successfully covered with medial hemisoleus, lateral hemisoleus, or peroneus brevis muscle flaps combined with split-thickness skin grafts, concentrated bone marrow aspirate, and platelet-rich plasma. All flaps healed with an average time to fixator removal of 8.3 weeks; there was 1 above-knee amputation that occurred approximately after successful wound closing and fixator removal. The combinatorial approach described here including several regenerative medicine tools is an effective means of lower limb reconstruction to avoid amputation.

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.

Microvascular Fragment Transplantation Improves Rat Dorsal Skin Flap Survival.

BACKGROUND: The development of flap necrosis distally remains a concern during microsurgical flap transfers because, at least in part, of decreased perfusion. Microvascular fragments (MVFs) are microvessels isolated from adipose tissue that are capable of improving tissue perfusion in a variety of tissue defects. The aim of this study was to determine whether the transplantation of MVFs in a dorsal rat skin flap model can improve flap survival. METHODS: A 10 × 3 cm flap was raised in a cranial to caudal fashion on the dorsal side of 16 Lewis rats, with the caudal side remaining intact. The rats were equally divided into a treatment group (MVFs) and a control group (sterile saline). At the time of surgery, sterile saline with or without MVFs was injected directly into the flap. Microvessel density was determined after harvesting flap tissue by counting vessels that positively stained for Griffonia simplicifolia lectin I-isolectin B4. Laser Doppler was used to measure blood flow before and after surgery and 7 and 14 days later. Flap survival was evaluated 7 and 14 days after surgery by evaluating the percentage of viable tissue of the flap with photodigital planimetry. RESULTS: Despite the lack of a significant difference in microvessel density and tissue perfusion, flap survival increased 6.4% (P < 0.05) in MVF-treated animals compared with controls. CONCLUSIONS: The use of MVFs may be a means to improve flap survival. Future studies are required to delineate mechanisms whereby this occurs and to further optimize their application.