Biomaterial and stem cell‐based strategies for skeletal muscle regeneration

Adult skeletal muscle can regenerate effectively after mild physical or chemical insult. Muscle trauma or disease can overwhelm this innate capacity for regeneration and result in heightened inflammation and fibrotic tissue deposition resulting in loss of structure and function. Recent studies have focused on biomaterial and stem cell‐based therapies to promote skeletal muscle regeneration following injury and disease. Many stem cell populations besides satellite cells are implicated in muscle regeneration. These stem cells include but are not limited to mesenchymal stem cells, adipose‐derived stem cells, hematopoietic stem cells, pericytes, fibroadipogenic progenitors, side population cells, and CD133⁺ stem cells. However, several challenges associated with their isolation, availability, delivery, survival, engraftment, and differentiation have been reported in recent studies. While acellular scaffolds offer a relatively safe and potentially off‐the‐shelf solution to cell‐based therapies, they are often unable to stimulate host cell migration and activity to a level that would result in clinically meaningful regeneration of traumatized muscle. Combining stem cells and biomaterials may offer a viable therapeutic strategy that may overcome the limitations associated with these therapies when they are used in isolation. In this article, we review the stem cell populations that can stimulate muscle regeneration in vitro and in vivo. We also discuss the regenerative potential of combination therapies that utilize both stem cell and biomaterials for the treatment of skeletal muscle injury and disease. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1246–1262, 2019.     

Effect of ultraviolet radiation—induced inflammation on epidermal wound healing

To examine the influence of ultraviolet radiation, a potent inducer of interleukin-1 and other growth factors, on the rate of epidermal migration, we used a porcine model of wound healing. Wounds were treated in one of the following treatment groups: (1) two minimal erythema doses of ultraviolet radiation once daily for 2 days before wounding, (2) two minimal erythema doses of ultraviolet radiation once daily for 2 days before wounding followed with continuous treatment until all wounds were 100% epithelized, (3) or no ultraviolet radiation treatment until healing was complete. Using a macroscopic salt-split technique, we examined epidermal specimens macroscopically for epithelialization. Treatment both before and after ultraviolet radiation significantly enhanced epithelialization when compared with non-ultraviolet radiation-treated control wounds. These experiments show that ultraviolet radiation treatment can augment the rate of healing of partial-thickness wounds.     

Validation of a laser‐assisted wound measurement device in a wound healing model

In the treatment and monitoring of a diabetic or chronic wound, accurate and repeatable measurement of the wound provides indispensable data for the patient's medical record. This study aims to measure the accuracy of the laser‐assisted wound measurement (LAWM) device against traditional methods in the measurement of area, depth and volume. We measured four ‘healing’ wounds in a Play‐Doh^®‐based model over five subsequent states of wound healing progression in which the model was irregularly filled in to replicate the healing process. We evaluated the LAWM device against traditional methods including digital photograph assessment with National Institutes of Health ImageJ software, measurements of depth with a ruler and weight‐to‐volume assessment with dental paste. Statistical analyses included analysis of variance (ANOVA) and paired t‐tests. We demonstrate that there are significantly different and nearly statistically significant differences between traditional ruler depth measurement and LAWM device measurement, but there are no statistically significant differences in area measurement. Volume measurements were found to be significantly different in two of the wounds. Rate of percentage change was analysed for volume and depth in the wound healing model, and the LAWM device was not significantly different than the traditional measurement technique. While occasionally inaccurate in its absolute measurement, the LAWM device is a useful tool in the clinician's arsenal as it reliably measures rate of percentage change in depth and volume and offers a potentially aseptic alternative to traditional measurement techniques.     

Evaluation of a starch‐based double layer scaffold for bone regeneration in a rat model

Damages in the maxillofacial bones are frequent in humans following trauma, metabolic diseases, neoplasia, or inflammatory processes. Many of the available treatments to regenerate bone are often ineffective. The goal of this work was to assess the in vivo behavior of an innovative double‐layered scaffold based on a blend of starch and polycaprolactone (SPCL) that comprises a membrane obtained by solvent casting, which aims to act as a guided tissue regeneration membrane, and a wet‐spun fiber mesh (in some cases functionalized with osteoconductive silanol groups) targeting bone regeneration. The behavior of the double layer scaffold, functionalized with silanol groups (SPCL‐Si) or without (SPCL), was assessed in a mandibular rodent model and compared to a commercial collagen membrane (positive control) and to empty defects (negative control). After 8 weeks of implantation, the micro‐computed tomography and the histomorphometric analysis revealed that the SPCL‐Si scaffolds induced significantly higher new bone formation compared to the collagen membrane and to the empty defects, although they had a similar performance when compared to the SPCL scaffolds. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:904–909, 2014.     

Mesenchymal stem cell‐conditioned medium accelerates wound healing with fewer scars

Mesenchymal stem cells (MSCs) derived from umbilical cord s (UC‐MSCs) have been shown to enhance cutaneous wound healing by means of the paracrine activity. Fibroblasts are the primary cells involved in wound repair. The paracrine effects of UC‐MSCs on dermal fibroblasts have not been fully explored in vitro or in vivo. Dermal fibroblasts were treated with conditioned media from UC‐MSCs (UC‐MSC‐CM). In this model, UC‐MSC‐CM increased the proliferation and migration of dermal fibroblasts. Moreover, adult dermal fibroblasts transitioned into a phenotype with a low myofibroblast formation capacity, a decreased ratio of transforming growth factor‐β1,3 (TGF‐β1/3) and an increased ratio of matrix metalloproteinase/tissue inhibitor of metalloproteinases (MMP/TIMP). Additionally, UC‐MSC‐CM‐treated wounds showed accelerated healing with fewer scars compared with control groups. These observations suggest that UC‐MSC‐CM may be a feasible strategy to promote cutaneous repair and a potential means to realise scarless healing.     

Improving translation success of cell‐based therapies in orthopaedics

There is a clear discrepancy between the growth of cell therapy and tissue engineering research in orthopaedics over the last two decades and the number of approved clinical therapies and products available to patients. At the 2015 annual meeting of the Orthopaedic Research Society, a workshop was held to highlight important considerations from the perspectives of an academic scientist, clinical researcher, and industry representative with the aim of helping researchers to successfully translate their ideas into clinical and commercial reality. Survey data acquired from workshop participants indicated an overall positive opinion on the future potential of cell‐based therapies to make a significant contribution to orthopaedic medicine. The survey also indicated an agreement on areas requiring improvement in the development of new therapies, specifically; increased support for fundamental research and education and improved transparency of regulatory processes. This perspectives article summarises the content and conclusions of the workshop and puts forward suggestions on how translational success of cell‐based therapies in orthopaedics may be achieved. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:17–21, 2016.     

New model of liver regeneration induced through use of vascular endothelial growth factor

INTRODUCTION: Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen. The objective of this study was to verify the proregenerative effects of VEGF in an experimental model of acute liver failure. MATERIALS AND METHODS: Sixty four rats that underwent intraperitoneal injection of carbon tetrachloride (CCl(4)) were randomly divided into two groups: group B animals received intravenous injection of VEGF(164) 1 hour following CCl(4) poisoning. Group A hosts were untreated. To obtain daily liver function tests (LFTs) and histological samples, on each day up to 8 days we sacrificed four rats in each group. RESULTS: The laboratory examinations showed notable alteration of LFTs in group A, while group B revealed only slight changes. The histological examination showed greater liver damage in group A compared with group B. CONCLUSION: Our results suggest that administration of exogenous VEGF protects the liver from CCl(4)-induced acute hepatic failure. Further studies are underway to assess whether exogenous VEGF is effective in other liver injuries.     

A surfactant‐based wound dressing can reduce bacterial biofilms in a porcine skin explant model

Bacterial biofilms have been found in many, if not all, chronic wounds. Their excessive extracellular matrix secretion and the metabolic changes that they undergo render them highly tolerant of many antibiotic and antimicrobial treatments. Physical removal and/or disruption are a common approach to treating wounds suspected of having bacterial biofilms. While many of these techniques use mechanical energy as the primary means of removal, we have begun to investigate if surfactants could facilitate the removal of bacterial biofilms, or if they might sensitise the biofilms to antimicrobial interventions. We tested a new surfactant‐based wound gel on an ex vivo porcine skin explant model infected with a functionally tolerant 3‐day biofilm. The wounds were dressed with a surfactant‐based gel directly on the wound or with moistened gauze. The wounds were then wiped daily with moistened gauze, and the gel or gauze was re‐applied. Each day, an explant from each group was harvested and tested for total viable bacteria counts and viable biofilm‐protected bacteria counts. The results show that daily wiping with moistened gauze led to an initial decrease of bacteria, but by day 3, the biofilm had been fully re‐established to the same level prior to the beginning of treatment. For the surfactant‐based treatment, there was no detectable functional biofilm after the first treatment. The gauze control, which was also subjected to daily wiping, still contained functional biofilms, indicating that this result was not due to wiping alone. The total bacteria in the surfactant‐treated explants steadily decreased through day 3, when there were no detectable bacteria, while the wiping‐only control bacteria counts remained steady. The use of a moist gauze to wipe the visually apparent slime off of a wound appears to be insufficient to reduce biofilm over a 3‐day period. Daily application of the surfactant gel dressing and wiping reduced the biofilm to undetectable levels within 3 days in a skin explant model. A 3‐day regimen of dressing the wound model with a surfactant gel followed by gentle removal of the gel by wiping with a moistened gauze appears to be a simple and adequate approach to removing a bacterial biofilm infection in an ex vivo model. Additional clinical evidence is needed to determine if this promising approach can perform the same in clinically infected chronic wounds.     

Embryonic stem cell–derived M2‐like macrophages delay cutaneous wound healing

In adults, repair of deeply injured skin wounds results in the formation of scar tissue, whereas in embryos wounds heal almost scar‐free. Macrophages are important mediators of wound healing and secrete cytokines and tissue remodeling enzymes. In contrast to host defense mediated by inflammatory M1 macrophages, wound healing and tissue repair involve regulatory M2/M2‐like macrophages. Embryonic/fetal macrophages are M2‐like, and this may promote scar‐free wound healing. In the present study, we asked whether atopical application of ex vivo generated, embryonic stem cell–derived macrophages (ESDM) improve wound healing in mice. ESDM were tested side by side with bone marrow–derived macrophages (BMDM). Compared to BMDM, ESDM resembled a less inflammatory and more M2‐like macrophage subtype as indicated by their reduced responsiveness to lipopolysaccharide, reduced expression of Toll‐like receptors, and reduced bacterial phagocytosis. Despite this anti‐inflammatory phenotype in cell culture, ESDM prolonged the healing of deep skin wounds even more than BMDM. Healed wounds had more scar formation compared to wounds receiving BMDM or cell‐free treatment. Our data indicate that atopical application of ex vivo generated macrophages is not a suitable cell therapy of dermal wounds.     

The comparison of two negative‐pressure wound therapy systems in a porcine model of wound healing

The purpose of this study was to compare two negative‐pressure wound healing systems (NPWT), ?75 mmHg with a silicone‐coated (SC) dressing and ?125 mmHg with polyurethane foam dressing (standard of care). In addition, this study compared the effects of two different dressing interfaces, SC dressing and gauze, with ?75 mmHg pressure. For both comparisons, two groups of five pigs were evaluated over a 21‐day time course. Two excisional wounds were made on each animal and NPWT dressings were applied. A canvas saddle was constructed to hold the NPWT device so the animal had free range of the pen. Dressings were changed twice a week and wound measurements were taken. Specimens for histology and gene expression analyses were taken on day 7 and 21. These data show that there is increased expression in a few genes associated with remodeling and inflammatory processes in the NPWT‐125 with polyurethane foam as compared with the NPWT‐75 with SC dressing. These two systems, however, are equivalent with respect to wound healing, histology, and gene expression over 21 days of healing. Further, we demonstrate that there is no difference in measure of healing between the SC dressing and a basic gauze dressing.     

Diabetic wound healing in a MMP9‐/‐ mouse model

Reduced mobilization of endothelial progenitor cells (EPCs) from the bone marrow (BM) and impaired EPC recruitment into the wound represent a fundamental deficiency in the chronic ulcers. However, mechanistic understanding of the role of BM‐derived EPCs in cutaneous wound neovascularization and healing remains incomplete, which impedes development of EPC‐based wound healing therapies. The objective of this study was to determine the role of EPCs in wound neovascularization and healing both under normal conditions and using single deficiency (EPC) or double‐deficiency (EPC + diabetes) models of wound healing. MMP9 knockout (MMP9 KO) mouse model was utilized, where impaired EPC mobilization can be rescued by stem cell factor (SCF). The hypotheses were: (1) MMP9 KO mice exhibit impaired wound neovascularization and healing, which are further exacerbated with diabetes; (2) these impairments can be rescued by SCF administration. Full‐thickness excisional wounds with silicone splints to minimize contraction were created on MMP9 KO mice with/without streptozotocin‐induced diabetes in the presence or absence of tail‐vein injected SCF. Wound morphology, vascularization, inflammation, and EPC mobilization and recruitment were quantified at day 7 postwounding. Results demonstrate no difference in wound closure and granulation tissue area between any groups. MMP9 deficiency significantly impairs wound neovascularization, increases inflammation, decreases collagen deposition, and decreases peripheral blood EPC (pb‐EPC) counts when compared with wild‐type (WT). Diabetes further increases inflammation, but does not cause further impairment in vascularization, as compared with MMP9 KO group. SCF improves neovascularization and increases EPCs to WT levels (both nondiabetic and diabetic MMP9 KO groups), while exacerbating inflammation in all groups. SCF rescues EPC‐deficiency and impaired wound neovascularization in both diabetic and nondiabetic MMP9 KO mice. Overall, the results demonstrate that BM‐derived EPCs play a significant role during wound neovascularization and that the SCF‐based therapy with controlled inflammation could be a viable approach to enhance healing in chronic diabetic wounds.     

Efficacy of a surfactant‐based wound dressing on biofilm control

The aim of this study was to evaluate the efficacy of both a nonantimicrobial and antimicrobial (1% silver sulfadiazine—SSD) surfactant‐based wound dressing in the control of Pseudomonas aeruginosa, Enterococcus sp, Staphylococcus epidermidis, Staphylococcus aureus, and methicillin‐resistant S. aureus (MRSA) biofilms. Anti‐biofilm efficacy was evaluated in numerous adapted American Standards for Testing and Materials (ASTM) standard biofilm models and other bespoke biofilm models. The ASTM standard models employed included the Minimum biofilm eradication concentration (MBEC) biofilm model (ASTM E2799) and the Centers for Disease Control (CDC) biofilm reactor model (ASTM 2871). Such bespoke biofilm models included the filter biofilm model and the chamberslide biofilm model. Results showed complete kill of microorganisms within a biofilm using the antimicrobial surfactant‐based wound dressing. Interestingly, the nonantimicrobial surfactant‐based dressing could disrupt existing biofilms by causing biofilm detachment. Prior to biofilm detachment, we demonstrated, using confocal laser scanning microscopy (CLSM), the dispersive effect of the nonantimicrobial surfactant‐based wound dressing on the biofilm within 10 minutes of treatment. Furthermore, the non‐antimicrobial surfactant‐based wound dressing caused an increase in microbial flocculation/aggregation, important for microbial concentration. In conclusion, this nonantimicrobial surfactant‐based wound dressing leads to the effective detachment and dispersion of in vitro biofilms. The use of surfactant‐based wound dressings in a clinical setting may help to disrupt existing biofilm from wound tissue and may increase the action of antimicrobial treatment.     

Characterisation of impaired wound healing in a preclinical model of induced diabetes using wide‐field imaging and conventional immunohistochemistry assays

Major complications of diabetes lead to inflammation and oxidative stress, delayed wound healing, and persistent ulcers. The high morbidity, mortality rate, and associated costs of management suggest a need for non‐invasive methods that will enable the early detection of at‐risk tissue. We have compared the wound‐healing process that occurs in streptozotocin (STZ)‐treated diabetic rats with non‐diabetic controls using contrast changes in colour photography (ie, Weber Contrast) and the non‐invasive optical method Spatial Frequency Domain Imaging (SFDI). This technology can be used to quantify the structural and metabolic properties of in‐vivo tissue by measuring oxyhaemoglobin concentration (HbO₂), deoxyhaemoglobin concentration (Hb), and oxygen saturation (StO₂) within the visible boundaries of each wound. We also evaluated the changes in inducible nitric oxide synthase (iNOS) in the dermis using immunohistochemistry. Contrast changes in colour photographs showed that diabetic rats healed at a slower rate in comparison with non‐diabetic control, with the most significant change occurring at 7 days after the punch biopsy. We observed lower HbO₂, StO₂, and elevated Hb concentrations in the diabetic wounds. The iNOS level was higher in the dermis of the diabetic rats compared with the non‐diabetic rats. Our results showed that, in diabetes, there is higher level of iNOS that can lead to an observed reduction in HbO₂ levels. iNOS is linked to increased inflammation, leading to prolonged wound healing. Our results suggest that SFDI has potential as a non‐invasive assessment of markers of wound‐healing impairment.     

Effect of cell‐based VEGF gene therapy on healing of a segmental bone defect

Fracture healing requires coordinated coupling between osteogenesis and angiogenesis in which vascular endothelial growth factor (VEGF) plays a key role. We hypothesized that targeted over‐expression of angiogenic and osteogenic factors within the fracture would promote bone healing by inducing development of new blood vessels and stimulating/affecting proliferation, survival, and activity of skeletal cells. Using a cell‐based method of gene transfer, without viral vector, 5.0 × 10⁶ fibroblasts transfected with VEGF were delivered to a 10‐mm bone defect in rabbit tibiae (Group 1) (n = 9); control groups were treated with fibroblasts (Group 2) (n = 7), or saline (Group 3) (n = 7) only. After 12 weeks, eight tibial fractures healed in Group 1, compared to four each in Groups 2 and 3. In Group 1, ossification was seen across the entire defect; in Groups 2 and 3, the defects were fibrous and sparsely ossified. Group 1 had more positively stained (CD31) vessels than Groups 2 and 3. MicroCT 3‐D showed complete bridging of the new bone for Group 1, but incomplete healing for Groups 2 and 3. MicroCT bone structural parameters showed significant differences between VEGF treatment and control groups (p < 0.05). These results indicate that the cell‐based VEGF gene therapy has significant angiogenic and osteogenic effects to enhance healing of a segmental defect in the long bone of rabbits. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:8–14, 2009     

Effectiveness of biofilm‐based wound care system on wound healing in chronic wounds

A biofilm plays a crucial role in delaying wound healing. Sharp debridement, a possible effective method for eliminating biofilms, can only be applied to the wound with visible necrotic tissue; thus, no option has been available for eliminating biofilms that are not accompanied by necrotic tissue. Wound blotting was recently developed to visualize biofilm noninvasively and quickly, and ultrasonic debridement is available for biofilm removal. Therefore, the purpose of this study was to investigate the efficacy of “biofilm‐based wound care system (BWCS),” a combination of wound blotting as a point‐of‐care testing and ultrasonic debridement, for promoting wound healing. Firstly, the cross‐sectional study was conducted to examine the proportion of biofilm removal by ultrasonic debridement in pressure ulcers [Study 1]. Subsequently, the retrospective cohort study was conducted to examine the effectiveness of BWCS for healing of chronic wounds [Study 2]. The proportions of wound healing between wounds treated with BWCS and those with standard care in the home‐visiting clinic were compared by Kaplan–Meier curve, and the Cox proportional hazard modeling was used to assess the effect of BWCS on wound healing. In Study 1, the median of biofilm removal proportion was 38.9% (interquartile range, 12.9–68.0%) for pressure ulcers treated with standard care and 65.2% (41.1–78.8%) for those treated with ultrasonic debridement (p = 0.009). In Study 2, the proportion of wound healing within 90 days was significantly higher in wounds treated with BWCS than in those treated with standard care (p = 0.001). The adjusted hazard ratio of BWCS for wound healing was 4.5 (95% confidence interval, 1.3–15.0; p = 0.015). In conclusion, we demonstrated that our novel approach, BWCS, can be a promising therapeutic strategy for visualizing biofilms that are not accompanied by necrotic tissue and promoting healing in chronic wounds.     

Effect of hyperoxia on vascular endothelial growth factor levels in a wound model

HYPOTHESIS: Hyperbaric oxygen (HBO) therapy increases vascular endothelial growth factor (VEGF) levels in wounds. DESIGN: Wounds were monitored for oxygen delivery during HBO treatment, and wound fluids were analyzed for VEGF and lactate on days 2, 5, and 10 following wounding. SETTING: Experimental animal model. INTERVENTIONS: Rats were randomized to HBO therapy and control groups. The HBO therapy was administered for 90 minutes, twice daily with 100% oxygen at 2.1 atmospheres absolute. Treatment was administered for 7 days following wounding. MAIN OUTCOME MEASURES: Vascular endothelial growth factor, PO(2), and lactate levels in wound fluid were measured on days 2, 5, and 10. RESULTS: Wound oxygen rises with HBO from nearly 0 mm Hg to as high as 600 mm Hg. The peak level occurs at the end of the 90-minute treatment, and hyperoxia of lessening degree persists for approximately 1 hour. The VEGF levels significantly increase with HBO by approximately 40% 5 days following wounding and decrease to control levels 3 days after exposures are stopped. Wound lactate levels remain unchanged with HBO treatment (range, 2.0-10.5 mmol/L). CONCLUSIONS: Increased VEGF production seems to explain in part the angiogenic action of HBO. This supports other data that hypoxia is not necessarily a requirement for wound VEGF production.