Biological attributes required for epidermal regeneration: Evaluation of the next-generation autologous cell harvesting device.

Early wound intervention and closure is critical for reducing infection and improving aesthetic and functional outcomes for patients with acute burn wounds and nonthermal full-thickness skin defects. Treatment of partial-thickness burns or full-thickness injuries with autologous skin cell suspension (ASCS) achieves robust wound closure while limiting the amount of donor skin compared with standard autografting. A Next Generation Autologous Cell Harvesting Device (NG-ACHD) was developed to standardize the preparation process for ASCS to ensure biological attributes are obtained known to correlate with well-established safety and performance data. This study compared ASCS prepared using the NG-ACHD and ACHD following the manufacturer's guidance, evaluating cellular yields, viability, apoptotic activity, aggregates, phenotypes and functional capacity. Non-inferiority was established for all biological attributes tested and comparable healing trajectories were demonstrated using an in vitro skin regeneration model. In addition to standardization, the NG-ACHD also provides workflow efficiencies with the potential to decrease training requirements and increase the ease of incorporation and utilization of ASCS in clinical practice.

A Micro-scale Humanized Ventilator-on-a-Chip to Examine the Injurious Effects of Mechanical Ventilation.

Patients with compromised respiratory function frequently require mechanical ventilation to survive. Unfortunately, non-uniform ventilation of injured lungs generates complex mechanical forces that lead to ventilator induced lung injury (VILI). Although investigators have developed lung-on-a-chip systems to simulate normal respiration, modeling the complex mechanics of VILI as well as the subsequent recovery phase is a challenge. Here we present a novel humanized in vitro ventilator-on-a-chip (VOC) model of the lung microenvironment that simulates the different types of injurious forces generated in the lung during mechanical ventilation. We used transepithelial/endothelial electrical resistance (TEER) measurements to investigate how individual and simultaneous application of the different mechanical forces alters real-time changes in barrier integrity during and after injury. We find that compressive stress (i.e. barotrauma) does not significantly alter barrier integrity while over-distention (20% cyclic radial strain, volutrauma) results in decreased barrier integrity that quickly recovers upon removal of mechanical stress. Conversely, surface tension forces generated during airway reopening (atelectrauma), result in a rapid loss of barrier integrity with a delayed recovery relative to volutrauma. Simultaneous application of cyclic stretching (volutrauma) and airway reopening (atelectrauma), indicate that the surface tension forces associated with reopening fluid-occluded lung regions is the primary driver of barrier disruption. Thus, our novel VOC system can monitor the effects of different types of injurious forces on barrier disruption and recovery in real-time and can be used to identify the biomechanical mechanisms of VILI.

Safety of bempedoic acid in patients at high cardiovascular risk and with statin intolerance.

BACKGROUND: Bempedoic acid is an oral adenosine triphosphate citrate lyase (ACL) inhibitor that lowers low-density lipoprotein cholesterol (LDL-C) blood levels. The Cholesterol Lowering via Bempedoic acid, an ACL-Inhibiting Regimen (CLEAR) Outcomes study demonstrated that bempedoic acid reduced cardiovascular (CV) risk in patients at high risk for CV events who were unwilling or unable to take guideline-recommended doses of statins. OBJECTIVE: To describe detailed safety information from CLEAR Outcomes, including events in the United States (US) prescribing information based on previous phase 3 hyperlipidemia studies. METHODS: CLEAR Outcomes was a double-blind trial conducted in 13,970 patients randomized to oral bempedoic acid 180 mg daily or placebo and followed for a median of 3.4 years. RESULTS: In patients who received at least one dose (7,001 bempedoic acid, 6,964 placebo), treatment emergent adverse events (AE) occurred in 86.3 % and 85 % of patients, respectively. COVID-19 was the most frequently reported AE in both groups. Changes in serum creatinine, blood urea nitrogen, hemoglobin, aminotransaminases, and uric acid were consistent with the known safety profile of bempedoic acid. Gout or gouty arthritis occurred in 3.2 % of bempedoic acid and 2.2 % of placebo patients. AE associated with tendinopathies, including tendon rupture, occurred in 2 % of patients in both treatment groups. Cholelithiasis occurred in 2.2 % of bempedoic acid and 1.2 % of placebo patients; AE related to gallbladder disease were similar between treatment groups. CONCLUSIONS: Bempedoic acid was well-tolerated compared with placebo. Safety data from the long-term CLEAR Outcomes study reinforce the positive benefit-risk profile of bempedoic acid.

Engineered Extracellular Vesicle-Based Therapies for Valvular Heart Disease.

Introduction: Valvular heart disease represents a significant burden to the healthcare system, with approximately 5 million cases diagnosed annually in the US. Among these cases, calcific aortic stenosis (CAS) stands out as the most prevalent form of valvular heart disease in the aging population.  CAS is characterized by the progressive calcification of the aortic valve leaflets, leading to valve stiffening. While aortic valve replacement is the standard of care for CAS patients, the long-term durability of prosthetic devices is poor, calling for innovative strategies to halt  or reverse disease progression. Here, we explor the potential use of novel extracellular vesicle (EV)-based nanocarriers for delivering molecular payloads to the affected valve tissue. This approach aims to reduce inflammation and potentially promote resorption of the calcified tissue. Methods: Engineered EVs loaded with the reprogramming myeloid transcription factors, CEBPA and Spi1, known to mediate the transdifferentiation of committed endothelial cells into macrophages. We evaluated the ability of these engineered EVs to deliver DNA and transcripts encoding CEBPA and Spil into calcified aortic valve tissue obtained from patients undergoing valve replacement due to aortic stenosis. We also investigated whether these EVs could induce the transdifferentiation of endothelial cells into macrophage-like cells. Results: Engineered EVs loaded with CEBPA + Spi1 were successfully derived from human dermal fibroblasts. Peak EV loading was found to be at 4 h after nanotransfection of donor cells.  These CEBPA + Spi1 loaded EVs effectively transfected aortic valve cells, resulting in the successful induction of transdifferentiation, both in vitro with  endothelial cells and ex vivo with valvular endothelial cells, leading to the development of anti-inflammatory macrophage-like cells. Conclusions: Our findings highlight the potential of engineered EVs as a next generation nanocarrier to target aberrant calcifications on diseased heart valves. This development holds promise as a novel therapy for high-risk patients who may not be suitable candidates for valve replacement surgery. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-023-00783-x.

Engineered Vasculogenic Extracellular Vesicles Drive Nonviral Direct Conversions of Human Dermal Fibroblasts into Induced Endothelial Cells and Improve Wound Closure.

Vasculogenic cell therapies have emerged as a powerful tool to increase vascularization and promote tissue repair/regeneration. Current approaches to cell therapies, however, rely mostly on progenitor cells, which pose significant risks (e.g., uncontrolled differentiation, tumorigenesis, and genetic/epigenetic abnormalities). Moreover, reprogramming methodologies used to generate induced endothelial cells (iECs) from induced pluripotent stem cells rely heavily on viral vectors, which pose additional translational limitations. This work describes the development of engineered human extracellular vesicles (EVs) capable of driving reprogramming-based vasculogenic therapies without the need for progenitor cells and/or viral vectors. The EVs were derived from primary human dermal fibroblasts (HDFs), and were engineered to pack transcription factor genes/transcripts of ETV2, FLI1, and FOXC2 (EFF). Our results indicate that in addition of EFF, the engineered EVs were also loaded with transcripts of angiogenic factors (e.g., VEGF-A, VEGF-KDR, FGF2). In vitro and in vivo studies indicate that such EVs effectively transfected HDFs and drove direct conversions towards iECs within 7-14 days. Finally, wound healing studies in mice indicate that engineered EVs lead to improved wound closure and vascularity. Altogether, our results show the potential of engineered human vasculogenic EVs to drive direct reprogramming processes of somatic cells towards iECs, and facilitate tissue repair/regeneration.

Laser Micropatterning Promotes Rete Ridge Formation and Enhanced Engineered Skin Strength without Increased Inflammation.

Rete ridges play multiple important roles in native skin tissue function, including enhancing skin strength, but they are largely absent from engineered tissue models and skin substitutes. Laser micropatterning of fibroblast-containing dermal templates prior to seeding of keratinocytes was shown to facilitate rete ridge development in engineered skin (ES) both in vitro and in vivo. However, it is unknown whether rete ridge development results exclusively from the microarchitectural features formed by ablative processing or whether laser treatment causes an inflammatory response that contributes to rete ridge formation. In this study, laser-micropatterned and non-laser- treated ES grafts were developed and assessed during culture and for four weeks post grafting onto full-thickness wounds in immunodeficient mice. Decreases in inflammatory cytokine secretion were initially observed in vitro in laser-treated grafts compared to non-treated controls, although cytokine levels were similar in both groups five days after laser treatment. Post grafting, rete ridge-containing ES showed a significant increase in vascularization at week 2, and in collagen deposition and biomechanics at weeks 2 and 4, compared with controls. No differences in inflammatory cytokine expression after grafting were observed between groups. The results suggest that laser micropatterning of ES to create rete ridges improves the mechanical properties of healed skin grafts without increasing inflammation.

A role for vitamin D and the vitamin D receptor in keloid disorder.

Keloids are disfiguring fibroproliferative lesions that can occur in susceptible individuals following any skin injury. They are extremely challenging to treat, with relatively low response rates to current therapies and high rates of recurrence after treatment. Although several distinct genetic loci have been associated with keloid formation in different populations, there has been no single causative gene yet identified and the molecular mechanisms guiding keloid development are incompletely understood. Further, although it is well known that keloids are more commonly observed in populations with dark skin pigmentation, the basis for increased keloid risk in skin of colour is not yet known. Because individuals with dark skin pigmentation are at higher risk for vitamin D deficiency, the role of vitamin D in keloid pathology has gained interest in the keloid research community. A limited number of studies have found lower serum vitamin D levels in patients with keloids, and reduced expression of the vitamin D receptor (VDR) in keloid lesions compared with uninjured skin. Vitamin D has documented anti-inflammatory, anti-proliferative and pro-differentiation activities, suggesting it may have a therapeutic role in suppression of keloid fibrosis. Here we review the evidence supporting a role for vitamin D and VDR in keloid pathology.

Injectable pulverized electrospun poly(lactic-co-glycolic acid) fibers improve human adipose tissue engraftment and volume retention.

Autologous adipose tissue is commonly used for tissue engraftment for the purposes of soft tissue reconstruction due to its relative abundance in the human body and ease of acquisition using liposuction methods. This has led to the adoption of autologous adipose engraftment procedures that allow for the injection of adipose tissues to be used as a "filler" for correcting cosmetic defects and deformities in soft tissues. However, the clinical use of such methods has several limitations, including high resorption rates and poor cell survivability, which lead to low graft volume retention and inconsistent outcomes. Here, we describe a novel application of milled electrospun poly(lactic-co-glycolic acid) (PLGA) fibers, which can be co-injected with adipose tissue to improve engraftment outcomes. These PLGA fibers had no significant negative impact on the viability of adipocytes in vitro and did not elicit long-term proinflammatory responses in vivo. Furthermore, co-delivery of human adipose tissue with pulverized electrospun PLGA fibers led to significant improvements in reperfusion, vascularity, and retention of graft volume compared to injections of adipose tissue alone. Taken together, the use of milled electrospun fibers to enhance autologous adipose engraftment techniques represents a novel approach for improving upon the shortcomings of such methods.

Scar Management and Dyschromia: A Summary Report from the 2021 American Burn Association State of the Science Meeting.

Burn scars, and in particular, hypertrophic scars, are a challenging yet common outcome for survivors of burn injuries. In 2021, the American Burn Association brought together experts in burn care and research to discuss critical topics related to burns, including burn scars, at its State of the Science conference. Clinicians and researchers with burn scar expertise, as well as burn patients, industry representatives, and other interested stakeholders met to discuss issues related to burn scars and discuss priorities for future burn scar research. The various preventative strategies and treatment modalities currently utilized for burn scars were discussed, including relatively noninvasive therapies such as massage, compression, and silicone sheeting, as well as medical interventions such as corticosteroid injection and laser therapies. A common theme that emerged is that the efficacy of current therapies for specific patient populations is not clear, and further research is needed to improve upon these treatments and develop more effective strategies to suppress scar formation. This will necessitate quantitative analyses of outcomes and would benefit from creation of scar biobanks and shared data resources. In addition, outcomes of importance to patients, such as scar dyschromia, must be given greater attention by clinicians and researchers to improve overall quality of life in burn survivors. Herein we summarize the main topics of discussion from this meeting and offer recommendations for areas where further research and development are needed.

Engineered extracellular vesicles from human skin cells induce pro-ß-cell conversions in pancreatic ductal cells.

Direct nuclear reprogramming has the potential to enable the development of ß cell replacement therapies for diabetes that do not require the use of progenitor/stem cell populations. However, despite their promise, current approaches to ß cell-directed reprogramming rely heavily on the use of viral vectors. Here we explored the use of extracellular vesicles (EVs) derived from human dermal fibroblasts (HDFs) as novel non-viral carriers of endocrine cell-patterning transcription factors, to transfect and transdifferentiate pancreatic ductal epithelial cells (PDCs) into hormone-expressing cells. Electrotransfection of HDFs with expression plasmids for Pdx1, Ngn3, and MafA (PNM) led to the release of EVs loaded with PNM at the gene, mRNA, and protein level. Exposing PDC cultures to PNM-loaded EVs led to successful transfection and increased PNM expression in PDCs, which ultimately resulted in endocrine cell-directed conversions based on the expression of insulin/c-peptide, glucagon, and glucose transporter 2 (Glut2). These findings were further corroborated in vivo in a mouse model following intraductal injection of PNM- vs sham-loaded EVs. Collectively these findings suggest that dermal fibroblast-derived EVs could potentially serve as a powerful platform technology for the development and deployment of non-viral reprogramming-based cell therapies for insulin-dependent diabetes.

Mechanical strain induces ex vivo expansion of periosteum.

Segmental bone defects present complex clinical challenges. Nonunion, malunion, and infection are common sequalae of autogenous bone grafts, allografts, and synthetic bone implants due to poor incorporation with the patient's bone. The current project explores the osteogenic properties of periosteum to facilitate graft incorporation. As tissue area is a natural limitation of autografting, mechanical strain was implemented to expand the periosteum. Freshly harvested, porcine periosteum was strained at 5 and 10% per day for 10 days with non-strained and free-floating samples serving as controls. Total tissue size, viability and histologic examination revealed that strain increased area to a maximum of 1.6-fold in the 10% daily strain. No change in tissue anatomy or viability via MTT or Ki67 staining and quantification was observed among groups. The osteogenic potential of the mechanical expanded periosteum was then examined in vivo. Human cancellous allografts were wrapped with 10% per day strained, fresh, free-floating, or no porcine periosteum and implanted subcutaneously into female, athymic mice. Tissue was collected at 8- and 16-weeks. Gene expression analysis revealed a significant increase in alkaline phosphatase and osteocalcin in the fresh periosteum group at 8-weeks post implantation compared to all other groups. Values among all groups were similar at week 16. Additionally, histological assessment with H&E and Masson-Goldner Trichrome staining showed that all periosteal groups outperformed the non-periosteal allograft, with fresh periosteum demonstrating the highest levels of new tissue mineralization at the periosteum-bone interface. Overall, mechanical expansion of the periosteum can provide increased area for segmental healing via autograft strategies, though further studies are needed to explore culture methodology to optimize osteogenic potential.

Myofibroblasts Are Not Characteristic Features of Keloid Lesions.

Keloids are disfiguring, scar-like lesions that are challenging to treat, with low response rates to current interventions and frequent recurrence. It has been widely reported that keloids are characterized by myofibroblasts, specialized contractile fibroblasts that express alpha-smooth muscle actin (α-SMA). However, evidence supporting a role for myofibroblasts in keloid pathology is inconclusive, with conflicting reports in the literature. This complicates development of more effective therapies, as the benefit of interventions targeting myofibroblasts is unclear. This study was undertaken to determine whether myofibroblasts can be considered characteristic of keloids. Methods: Myofibroblasts in tissue sections from keloids, hypertrophic scars (HTSs), and normal skin were localized by α-SMA immunostaining. Expression of α-SMA mRNA (ACTA2 gene) in normal skin and keloid tissue, and in fibroblasts from normal skin, keloid, and HTSs, was measured using quantitative polymerase chain reaction. Results: Normal skin did not exhibit α-SMA-expressing myofibroblasts, but myofibroblasts were identified in 50% of keloids and 60% of HTSs. No significant differences in ACTA2 expression between keloid and normal skin tissue were observed. Mean ACTA2 expression was higher in HTS (2.54-fold, P = 0.005) and keloid fibroblasts (1.75-fold, P = 0.046) versus normal fibroblasts in vitro. However, α-SMA expression in keloids in vivo was not associated with elevated ACTA2 in keloid fibroblasts in vitro. Conclusions: Despite elevated ACTA2 in cultured keloid fibroblasts, myofibroblast presence is not a consistent feature of keloids. Therefore, therapies that target myofibroblasts may not be effective for all keloids. Further research is required to define the mechanisms driving keloid formation for development of more effective therapies.

Advantages and Disadvantages of Using Small and Large Animals in Burn Research: Proceedings of the 2021 Research Special Interest Group.

Multiple animal species and approaches have been used for modeling different aspects of burn care, with some strategies considered more appropriate or translatable than others. On April 15, 2021, the Research Special Interest Group of the American Burn Association held a virtual session as part of the agenda for the annual meeting. The session was set up as a pro/con debate on the use of small versus large animals for application to four important aspects of burn pathophysiology: burn healing/conversion, scarring, inhalation injury, and sepsis. For each of these topics, two experienced investigators (one each for small and large animal models) described the advantages and disadvantages of using these preclinical models. The use of swine as a large animal model was a common theme due to anatomic similarities with human skin. The exception to this was a well-defined ovine model of inhalation injury; both of these species have larger airways which allow for incorporation of clinical tools such as bronchoscopes. However, these models are expensive and demanding from labor and resource standpoints. Various strategies have been implemented to make the more inexpensive rodent models appropriate for answering specific questions of interest in burns. Moreover, modeling burn-sepsis in large animals has proven difficult. It was agreed that the use of both small and large animal models has merit for answering basic questions about the responses to burn injury. Expert opinion and the ensuing lively conversations are summarized herein, which we hope will help inform experimental design of future research.

Titanium as a Possible Modifier of Inflammation Around Dental Implants.

PURPOSE: The exact etiopathogenesis of peri-implant diseases remains unclear. While significant information on molecular markers is available, studies on biomarkers related to possible biocorrosion are sparse. This study aimed to evaluate periimplant crevicular fluid (PICF) for possible titanium (Ti) contamination and explore associations between clinical findings, inflammatory mediators, and Ti levels. MATERIALS AND METHODS: Patients with implant-supported restoration (≥ 1 year in function) were recruited for this cross-sectional study. Demographics, systemic, and periodontal health history were recorded. Clinical evaluations were conducted to reach peri-implant/periodontal diagnoses and grade severity of peri-implant soft tissue inflammation. Crevicular fluid (CF) was collected from both implants and adjacent teeth (PICF, gingival crevicular fluid [GCF]) and analyzed for Ti (inductively coupled plasma mass spectrometry) and inflammatory mediators (V-plex assays). Multiple regression analysis with a linear mixed effect model was used to analyze possible associations between clinical diagnosis, PICF/GCF cytokine, and Ti concentrations. RESULTS: Seventy-seven patients (aged 62 ± 2 years; 39 male) with 117 implants (9 ± 1 years in function) were recruited. Diabetes, positive periodontitis history, and current/former smoking were reported by 8%, 39%, and 39% of subjects, respectively. Seventy-nine implant sites (63 patients) were included in CF cytokine analysis, and 45 of these sites (42 patients) were paired with Ti analysis. Statistically significant increases from health to disease were noted in log-transformed PICF concentrations of IL-1ß, IL-6, IL-10, and INF-γ (P ≤ .05). Also, statistically significant increases from health to severe clinical inflammation were detected in log-transformed PICF concentrations of IL-8, IL-13, and TNF-α (P ≤ .05). Ti was detected in the majority (82%) of PICF and GCF samples. There was no statistically significant difference in log-transformed Ti concentration based on disease status. However, log-transformed Ti concentration was positively correlated to IL-1ß, IL-2, IL-4, IL-8, IL-13, and INF-γ concentrations when data were adjusted for site-specific health (P ≤ .05). CONCLUSION: Ti was detectable in PICF and adjacent GCF, even in health. Specific inflammatory mediator concentrations were increased in peri-implant disease and significantly associated with Ti concentrations, even when data were adjusted for peri-implant health status. Increased GCF inflammatory mediator concentrations were also associated with increased Ti concentrations. Ti effects on peri-implant as well as periodontal tissues require additional longitudinal investigations.

Isolation and feeder-free primary culture of four cell types from a single human skin sample.

Four types of primary cells-dermal fibroblasts, dermal microvascular endothelial cells, epidermal keratinocytes, and epidermal melanocytes-can be isolated simultaneously from a single human skin sample, without the use of xenogeneic murine feeder cells. This protocol describes the procedures for isolation of these cells from adult full-thickness skin obtained from surgical discard tissue. The cells isolated using this protocol contain stem cell populations and are competent to form functional skin tissue in three-dimensional reconstructed skin models. For complete details on the use and execution of this profile, please refer to Supp et al. (2002), Boyce et al. (2015), Boyce et al. (2017a), Boyce et al. (2017b), and Supp et al. (2019).

3D engineered human gingiva fabricated with electrospun collagen scaffolds provides a platform for in vitro analysis of gingival seal to abutment materials.

In order to advance models of human oral mucosa towards routine use, these models must faithfully mimic the native tissue structure while also being scalable and cost efficient. The goal of this study was to develop a low-cost, keratinized human gingival model with high fidelity to human attached gingiva and demonstrate its utility for studying the implant-tissue interface. Primary human gingival fibroblasts (HGF) and keratinocytes (HGK) were isolated from clinically healthy gingival biopsies. Four matrices, electrospun collagen (ES), decellularized dermis (DD), type I collagen gels (Gel) and released type I collagen gels (Gel-R)) were tested to engineer lamina propria and gingiva. HGF viability was similar in all matrices except for Gel-R, which was significantly decreased. Cell penetration was largely limited to the top layers of all matrices. Histomorphometrically, engineered human gingiva was found to have similar appearance to the native normal human gingiva except absence of rete pegs. Immunohistochemical staining for cell phenotype, differentiation and extracellular matrix composition and organization within 3D engineered gingiva made with electrospun collagen was mostly in agreement with normal gingival tissue staining. Additionally, five types of dental material posts (5-mm diameter x 3-mm height) with different surface characteristics were used [machined titanium, SLA (sandblasted-acid etched) titanium, TiN-coated (titanium nitride-coated) titanium, ceramic, and PEEK (Polyetheretherketone) to investigate peri-implant soft tissue attachment studied by histology and SEM. Engineered epithelial and stromal tissue migration to the implant-gingival tissue interface was observed in machined, SLA, ceramic, and PEEK groups, while TiN was lacking attachment. Taken together, the results suggest that electrospun collagen scaffolds provide a scalable, reproducible and cost-effective lamina propria and 3D engineered gingiva that can be used to explore biomaterial-soft tissue interface.

Mechanomodulation of Burn Scarring Via Pressure Therapy.

Significance: The physical and psychological sequalae of burn injuries account for 10 million disability-adjusted life years lost annually. Hypertrophic scarring (HSc) after burn injury results in reduced mobility, contracture, pain, itching, and aesthetic changes for burn survivors. Despite the prevalence of scarring and the number of scar therapies available, none are highly effective at preventing HSc after burn injury. Recent Advances: Recent studies modulating the mechanical environment surrounding incisional and excisional wounds have shown off-loading of tension to be a powerful strategy to prevent scar formation. Preclinical studies applying force perpendicular to the surface of the skin or using a combination of pressure both circumferentially and perpendicularly have shown substantial reductions in scar thickness and contraction after burn injury. Critical Issues: Though pressure therapy is highly effective in preclinical studies, outcomes in clinical studies have been variable and may be a result of differing therapy protocols and garment material fatigue. A recent adult clinical study reported a significant reduction in pressure after 1 month of use and significant reduction between 1 and 2 months of use, resulting in below therapeutic doses of pressure applied after only 1 month of use. Future Directions: To enhance efficacy of pressure garments, new low-fatigue materials must be developed for use in standard garments or garments must be redesigned to allow for adjustment to compensate for the loss of pressure with time. Additionally, measurements of applied pressure should be performed routinely during clinic visits to ensure that therapeutic doses of pressure are being delivered.

Collagen-Based Electrospun Materials for Tissue Engineering: A Systematic Review.

Collagen is a key component of the extracellular matrix (ECM) in organs and tissues throughout the body and is used for many tissue engineering applications. Electrospinning of collagen can produce scaffolds in a wide variety of shapes, fiber diameters and porosities to match that of the native ECM. This systematic review aims to pool data from available manuscripts on electrospun collagen and tissue engineering to provide insight into the connection between source material, solvent, crosslinking method and functional outcomes. D-banding was most often observed in electrospun collagen formed using collagen type I isolated from calfskin, often isolated within the laboratory, with short solution solubilization times. All physical and chemical methods of crosslinking utilized imparted resistance to degradation and increased strength. Cytotoxicity was observed at high concentrations of crosslinking agents and when abbreviated rinsing protocols were utilized. Collagen and collagen-based scaffolds were capable of forming engineered tissues in vitro and in vivo with high similarity to the native structures.