Mesenchymal stroma cells trigger early attraction of M1 macrophages and endothelial cells into fibrin hydrogels,stimulating long bone healing without long-term engraftment

Implantation of mesenchymal stroma cells (MSCs) is an attractive approach to stimulate closure of large bone defects but an optimal carrier has yet to be defined. MSCs may display trophic and/or immunomodulatory features or stimulate bone healing by their osteogenic activity. The aim of this study was to unravel whether fibrin hydrogel supports early actions of implanted MSCs, such as host cell recruitment, immunomodulation and tissue regeneration, in long bone defects. Female rats received cell-free fibrin or male MSCs embedded in a fibrin carrier into plate-stabilized femoral bone defects. Removed callus was analyzed for host cell invasion (day 6), local cytokine expression (days 3 and 6) and persistence of male MSCs (days 3, 6, 14 and 28). Fibrin–MSC composites triggered fast attraction of host cells into the hydrogel while cell-free fibrin implants were not invaded. A migration front dominated by M1 macrophages and endothelial progenitor cells formed while M2 macrophages remained sparse. Only MSC-seeded fibrin hydrogel stimulated early tissue maturation and primitive vessel formation at day 6 in line with significantly higher VEGF mRNA levels recorded at day 3. Local TNF-α, IL-1β and IL-10 expression indicated a balanced immune cell activity independent of MSC implantation. Implanted MSCs persisted until day 14 but not day 28. Our results demonstrate that fibrin hydrogel is an attractive carrier for MSC implantation into long bone defects, supporting host cell attraction and pro-angiogenic activity. By this angiogenesis, implant integration and tissue maturation was stimulated in long bone healing independent of long-term engraftment of implanted MSCs.     

Second-harmonic generation microscopy for assessment of mesenchymal stem cell-seeded acellular dermal matrix in wound-healing

Direct intra-skin injection of mesenchymal stem cells (MSCs) and the use of biomaterial scaffolds for grafts are both promising approaches of skin wound repair, however they still cannot generate skin that completely resembles the natural skin structures. In this study, we combined these two approaches by using acellular dermal matrix (ADM) recellularized with MSCs to repair cutaneous wounds in a murine model and two-photon fluorescence (TPF) microscopy and second-harmonic generation (SHG) microscopy to assess the effects of this therapy on wound healing. Bone marrow-derived mesenchymal stem cells (BM-MSCs) were tagged with GFP and seeded into ADM (ADM-MSC) via MSC and ADM co-culture. ADM-MSC, ADM or saline was applied to murine excisional skin wounds and wound-healing was evaluated by histological examination on days 7, 14, 21 and TFP microscopy on days 1, 3, 5 and 21 post-treatment. ADM-MSC promoted healing significantly more than treatment with ADM or saline alone, as it led to substantial neovascularization and complete skin appendage regeneration. Furthermore, the SHG microscopic imaging technique proved to be a useful tool for monitoring changes in the collagen network at the wound site during the healing process and assessing the effects of different therapies.     

Local and Remote Effects of Mesenchymal Stem Cell Administration on Skin Wound Regeneration

Wound healing is an important medical problem. We evaluated the efficacy of locally administered mesenchymal stem cells (MSCs) isolated from human umbilical cords on the dynamics of skin wound healing. The study was conducted on the backs of Wistar rats, where two square wounds were created by removing all layers of the skin. Four groups were studied in two series of experiments: (1) a Control_NaCl group (the wounds were injected with 0.9% NaCl solution) and a Control_0 group (intact wounds on the opposite side of the same rat’s back); (2) an MSC group (injected MSCs, local effect) and a Control_sc group (intact wounds on the opposite side of the back, remote MSC effect). The area and temperature of the wounds and the microcirculation of the wound edges were measured. Histological and morphometric studies were performed on days 3 and 7 after the wounds were created. The results showed that the injection trauma (Control_NaCl) slowed the regeneration process. In both MSC groups (unlike in either control group), we observed no increase in the area of the wounds; in addition, we observed inhibition of the inflammatory process and improved wound regeneration on days 1–3 in the remote group and days 1–5 in the local (injected) group. The MSC and Control_sc groups demonstrated improved microcirculation and suppression of leukocyte infiltration on day 3. On day 7, all the studied parameters of the wounds of the Control_0 group were the same as those of the wounds that received cell therapy, although in contrast to the results of the Control_ NaCl group, fibroblast proliferation was greater in the MSC and Control_sc groups. The dynamics of the size of the wounds were comparable for both local and remote application of MSCs. Thus, even a one-time application of MSCs was effective during the first 3–5 days after injury due to anti-inflammatory processes, which improved the regeneration process. Remote application of MSC, as opposed to direct injection, is advisable, especially in the case of multiple wounds, since the results were indistinguishable between the groups and injection trauma was shown to slow healing.     

Coculture of human mesenchymal stem cells and articular chondrocytes reduces hypertrophy and enhances functional properties of engineered cartilage

Mesenchymal stem cells (MSCs) are being recognized as a viable cell source for cartilage repair; however, it still remains a challenge to recapitulate the functional properties of native articular cartilage using only MSCs. Additionally, MSCs may exhibit a hypertrophic phenotype under chondrogenic induction, resulting in calcification after ectopic transplantation. With this in mind, the objective of this study was to assess whether the addition of chondrocytes to MSC cultures influences the properties of tissue-engineered cartilage and MSC hypertrophy when cultured in hyaluronic acid hydrogels. Mixed cell populations (human MSCs and human chondrocytes at a ratio of 4:1) were encapsulated in the hydrogels and exhibited significantly higher Young's moduli, dynamic moduli, glycosaminoglycan levels, and collagen content than did constructs seeded with only MSCs or chondrocytes. Furthermore, the deposition of collagen X, a marker of MSC hypertrophy, was significantly lower in the coculture constructs than in the constructs seeded with MSCs alone. When MSCs and chondrocytes were cultured in distinct gels, but in the same wells, there was no improvement in biomechanical and biochemical properties of the engineered tissue, implying that a close proximity is essential. This approach can be used to improve the properties and prevent calcification of engineered cartilage formed from MSC-seeded hydrogels with the addition of lower fractions of chondrocytes, leading to improved clinical outcomes.     

Grafting of mesenchymal stem cell-seeded small intestinal submucosa to repair the deep partial-thickness burns

Purpose: Regenerative medicine provides many treatments for burn wounds, of which cell-seeded substitutes are encouraging for large and deep burns. To assess the feasibility of mesenchymal stem cell (MSC)-seeded small intestinal submucosa (SIS) to repair the deep partial-thickness burns, a rat study was performed. Materials & Methods: The burn model was created by contacting the dorsal surface directly with boiled water for 10 seconds. MSCs at passage 3 were seeded on the SIS before implantation. Three days after burn injury, the grafts were implanted onto the burn area. At 3, 7, 14 and 21 days post implantation, gross observation and histological assessments were performed. Results: SIS alone and MSC-seeded SIS were able to accelerate the burn wound closure by enhancing granulation tissue formation, increasing wound maturity, improving revascularization, and inducing the proliferation of neo-epidermal cells. Additionally, MSC-seeded SIS was much more effective than SIS alone for the repair of deep partial-thickness burns. Conclusion: Both SIS and MSC-seeded SIS were able to repair the large and deep burn wounds and the loaded MSCs possessed positive effects to accelerate the wound closure in a rat model.     

The effect of hyaluronan-based delivery of stromal cell-derived factor-1 on the recruitment of MSCs in degenerating intervertebral discs

Intervertebral disc (IVD) degeneration is the leading cause of low back pain and disability in the active population. Transplantation of mesenchymal stem cells (MSCs) in a hydrogel carrier can induce regenerative effects in degenerated IVDs. Moreover, it was found that degenerative discs release chemoattractants effective in MSC recruitment. Based on these findings, we hypothesized that an injectable hydrogel that can enhance the number of migrated MSCs in the IVD and provide a suitable matrix for their survival and differentiation would be ideal. The purpose of this study was to evaluate the potential of a thermoreversible hyaluronan-poly(N-isopropylacrylamide) (HAP) hydrogel as chemoattractant delivery system to recruit human MSCs in degenerative IVDs. The results demonstrate that HAP hydrogels containing stromal cell derived factor-1 (SDF-1) significantly increased the number of MSCs migrating into nucleotomized discs compared with discs treated with only HAP or SDF-1 in solution. HAP hydrogels releasing SDF-1 enhanced both the number of recruited cells and their migration distance in the IVD tissue. Furthermore, this phenomenon was dependent on MSC donor age. In conclusion, HAP SDF-1 is effective for the recruitment of stem cells in the IVD, thus opening new possibilities for the development of regenerative therapies based on endogenous cell migration.     

Degradable hydrogels for spatiotemporal control of mesenchymal stem cells localized at decellularized bone allografts

The transplantation of cells, such as mesenchymal stem cells (MSCs), has numerous applications in the field of regenerative medicine. For cell transplantation strategies to be successful therapeutically, cellular localization and persistence must be controlled to maximize cell-mediated contributions to healing. Herein, we demonstrate that hydrolytic degradation of poly(ethylene glycol) (PEG) hydrogels can be used to spatiotemporally control encapsulated MSC localization to decellularized bone allografts, both in vitro and in vivo. By altering the number of hydrolytically degradable lactide repeat units within PEG-d,l-lactide-methacrylate macromers, a series of hydrogels was synthesized that degraded over ∼1, 2 and 3 weeks. MSCs were encapsulated within these hydrogels formed around decellularized bone allografts, and non-invasive, longitudinal fluorescence imaging was used to track cell persistence both in vitro and in vivo. Spatiotemporal localization of MSCs to the exterior of bone allograft surfaces was similar to in vitro hydrogel degradation kinetics despite hydrogel mesh sizes being ∼2–3 orders of magnitude smaller than MSC size throughout the degradation process. Thus, localized, cell-mediated degradation and MSC migration from the hydrogels are suspected, particularly as ∼10% of the total transplanted MSC population was shown to persist in close proximity (within ∼650 μm) to grafts 7 weeks after complete hydrogel degradation. This work demonstrates the therapeutic utility of PEG-based hydrogels for controlling spatiotemporal cell transplantation for a myriad of regenerative medicine strategies.     

Dynamic compressive loading enhances cartilage matrix synthesis and distribution and suppresses hypertrophy in hMSC-laden hyaluronic acid hydrogels

Mesenchymal stem cells (MSCs) are being recognized as a viable cell source for cartilage repair, and there is growing evidence that mechanical signals play a critical role in the regulation of stem cell chondrogenesis and in cartilage development. In this study we investigated the effect of dynamic compressive loading on chondrogenesis, the production and distribution of cartilage specific matrix, and the hypertrophic differentiation of human MSCs encapsulated in hyaluronic acid (HA) hydrogels during long term culture. After 70 days of culture, dynamic compressive loading increased the mechanical properties, as well as the glycosaminoglycan (GAG) and collagen contents of HA hydrogel constructs in a seeding density dependent manner. The impact of loading on HA hydrogel construct properties was delayed when applied to lower density (20 million MSCs/ml) compared to higher seeding density (60 million MSCs/ml) constructs. Furthermore, loading promoted a more uniform spatial distribution of cartilage matrix in HA hydrogels with both seeding densities, leading to significantly improved mechanical properties as compared to free swelling constructs. Using a previously developed in vitro hypertrophy model, dynamic compressive loading was also shown to significantly reduce the expression of hypertrophic markers by human MSCs and to suppress the degree of calcification in MSC-seeded HA hydrogels. Findings from this study highlight the importance of mechanical loading in stem cell based therapy for cartilage repair in improving neocartilage properties and in potentially maintaining the cartilage phenotype.     

Modulation of differentiation and mineralization of marrow stromal cells cultured on biomimetic hydrogels modified with Arg-Gly-Asp containing peptides

We synthesized biomimetic hydrogels modified with an osteopontin-derived peptide (ODP) and used them as a substrate for in vitro culture of marrow stromal cells (MSCs) to investigate the effect of the biomimetic surface on differentiation of MSCs into osteoblasts. Proliferation and biological assays for 16 days proved that MSCs became differentiated into osteoblasts secreting osteogenic phenotypic markers such as alkaline phosphatase (ALP), osteopontin, and mineralized calcium. In addition, there was an additive effect of the cell-binding peptide on differentiation and mineralization of MSCs cultured in the presence of soluble osteogenic supplements in cell culture media. For example, calcium content at day 16 on peptide-modified hydrogels was significantly higher than on tissue culture polystyrene. Two general trends were observed: (1) proliferation of MSCs decreased as the amount of differentiation markers increased, and (2) higher peptide concentrations accelerated the differentiation of MSCs. On the hydrogel modified with ODP, ALP activity exhibited a maximum value of 36.7 +/- 4.2 pmol/cell/h at day 10 for the concentration of 2 micromol/g while the culture time needed for maximum ALP activity occurred on day 13 for the lower concentrations. On the same hydrogel, the calcium content at day 10 was 21.4 +/- 2.3 ng/cell for the peptide concentration of 2 micromol/g and 1.0 +/- 0.3 ng/cell for 1.0 micromol/g. We used Gly-Arg-Gly-Asp-Ser (GRGDS) for modification of the hydrogel as a comparison to the results with ODP. However, osteoblast development was not significantly affected by the nature of the binding peptide sequences. These results suggest that MSC function can be modulated by variation of the peptide concentration in biomimetic hydrogels used for scaffold-based bone tissue engineering.     

Thiol-ene Michael-type formation of gelatin/poly(ethylene glycol) biomatrices for three-dimensional mesenchymal stromal/stem cell administration to cutaneous wounds

Mesenchymal stromal/stem cells (MSCs) are considered promising cellular therapeutics in the fields of tissue engineering and regenerative medicine. MSCs secrete high concentrations of immunomodulatory cytokines and growth factors, which exert paracrine effects on infiltrating immune and resident cells in the wound microenvironment that could favorably promote healing after acute injury. However, better spatial delivery and improved retention at the site of injury are two factors that could improve the clinical application of MSCs. In this study, we utilized thiol-ene Michael-type addition for rapid encapsulation of MSCs within a gelatin/poly(ethylene glycol) biomatrix. This biomatrix was also applied as a provisional dressing to full thickness wounds in Sprague–Dawley rats. The three-way interaction of MSCs, gelatin/poly(ethylene glycol) biomatrices, and host immune cells and adjacent resident cells in the wound microenvironment favorably modulated wound progression and host response. In this model we observed attenuated immune cell infiltration, lack of foreign giant cell (FBGC) formation, accelerated wound closure and re-epithelialization, as well as enhanced neovascularization and granulation tissue formation by 7 days. The MSC entrapped in the gelatin/poly(ethylene glycol) biomatrix localized cell presentation adjacent to the wound microenvironment and thus mediated the early resolution of inflammatory events and facilitated the proliferative phases in wound healing.     

Ultrashort peptide nanofibrous hydrogels for the acceleration of healing of burn wounds

There is an unmet clinical need for wound dressings to treat partial thickness burns that damage the epidermis and dermis. An ideal dressing needs to prevent infection, maintain skin hydration to facilitate debridement of the necrotic tissue, and provide cues to enhance tissue regeneration. We developed a class of ‘smart’ peptide hydrogels, which fulfill these criteria. Our ultrashort aliphatic peptides have an innate tendency to self-assemble into helical fibers, forming biomimetic hydrogel scaffolds which are non-immunogenic and non-cytotoxic. These nanofibrous hydrogels accelerated wound closure in a rat model for partial thickness burns. Two peptide hydrogel candidates demonstrate earlier onset and completion of autolytic debridement, compared to Mepitel^®, a silicone-coated polyamide net used as standard-of-care. They also promote epithelial and dermal regeneration in the absence of exogenous growth factors, achieving 86.2% and 92.9% wound closure respectively, after 14 days. In comparison, only 62.8% of the burnt area is healed for wounds dressed with Mepitel^®. Since the rate of wound closure is inversely correlated with hypertrophic scar formation and infection risks, our peptide hydrogel technology fills a niche neglected by current treatment options. The regenerative properties can be further enhanced by incorporation of bioactive moieties such as growth factors and cytokines.     

Immunomodulation by mesenchymal stem cells combats the foreign body response to cell-laden synthetic hydrogels

The implantation of non-biological materials, including scaffolds for tissue engineering, ubiquitously leads to a foreign body response (FBR). We recently reported that this response negatively impacts fibroblasts encapsulated within a synthetic hydrogel and in turn leads to a more severe FBR, suggesting a cross-talk between encapsulated cells and inflammatory cells. Given the promise of mesenchymal stem cells (MSCs) in tissue engineering and recent evidence of their immunomodulatory properties, we hypothesized that MSCs encapsulated within poly(ethylene glycol) (PEG) hydrogels will attenuate the FBR. In vitro, murine MSCs encapsulated within PEG hydrogels attenuated classically activated primary murine macrophages by reducing gene expression and protein secretion of pro-inflammatory cytokines, most notably tumor necrosis factor-α. Using a COX2 inhibitor, prostaglandin E2 (PGE2) was identified as a mediator of MSC immunomodulation of macrophages. In vivo, hydrogels laden with MSCs, osteogenically differentiating MSCs, or no cells were implanted subcutaneously into C57BL/6 mice for 28 days to assess the impact of MSCs on the fibrotic response of the FBR. The presence of encapsulated MSCs reduced fibrous capsule thickness compared to acellular hydrogels, but this effect diminished with osteogenic differentiation. The use of MSCs prior to differentiation in tissue engineering may therefore serve as a dynamic approach, through continuous cross-talk between MSCs and the inflammatory cells, to modulate macrophage activation and attenuate the FBR to implanted synthetic scaffolds thus improving the long-term tissue engineering outcome.     

Human mesenchymal stem cell differentiation to NP-like cells in chitosan-glycerophosphate hydrogels

Intervertebral disc (IVD) degeneration is one of the major causes of low back pain. As current clinical treatments are aimed at restoring biomechanical function and providing symptomatic relief, interest in methods focused on biological repair has increased. Several tissue engineering approaches using different cell types and hydrogels/scaffolds have been proposed. Owing to the unsuitable nature of degenerate cells for tissue engineering attention has focused on the use of mesenchymal stem cells (MSCs). Additionally, while rigid scaffolds have been demonstrated to allow MSC differentiation to the chondrocyte-like cells of the IVD, hydrogels are being increasingly studied as they allow minimally invasive implantation without extensive damage to the IVD. Here, we have studied the temperature-sensitive hydrogel chitosan-glycerophosphate (C/Gp), seeded with human MSCs and cultured for 4 weeks in standard medium. We have analysed the gene and protein expression profile of the MSCs and compared it to that of both nucleus pulposus (NP) cells and articular chondrocytes cultured in C/Gp. Gene expression analysis for chondrocytic-cell marker genes demonstrated differentiation of MSCs to a phenotype which showed similarities to both articular chondrocytes and NP cells. Conventional PCR demonstrated a lack of expression of osteogenic marker genes and the hypertrophic marker gene type X collagen. MSCs also secreted both proteoglycans and collagens in a ratio, which more closely resembled that of NP cells than articular chondrocytes. These results therefore suggest that MSC-seeded C/Gp gels could be used clinically for the regeneration of the degenerate human IVD.     

Autologous bone marrow-derived cultured mesenchymal stem cells delivered in a fibrin spray accelerate healing in murine and human cutaneous wounds

The nonhematopoietic component of bone marrow includes multipotent mesenchymal stem cells (MSC) capable of differentiating into fat, bone, muscle, cartilage, and endothelium. In this report, we describe the cell culture and characterization, delivery system, and successful use of topically applied autologous MSC to accelerate the healing of human and experimental murine wounds. A single bone marrow aspirate of 35-50 mL was obtained from patients with acute wounds (n = 5) from skin cancer surgery and from patients with chronic, long-standing, nonhealing lower extremity wounds (n = 8). Cells were grown in vitro under conditions favoring the propagation of MSC, and flow cytometry and immunostaining showed a profile (CD29+, CD44+, CD105+, CD166+, CD34-, CD45-) highly consistent with published reports of human MSC. Functional induction studies confirmed that the MSC could differentiate into bone, cartilage, and adipose tissue. The cultured autologous MSC were applied up to four times to the wounds using a fibrin polymer spray system with a double-barreled syringe. Both fibrinogen (containing the MSC) and thrombin were diluted to optimally deliver a polymerized gel that immediately adhered to the wound, without run-off, and yet allowing the MSC to remain viable and migrate from the gel. Sequential adjacent sections from biopsy specimens of the wound bed after MSC application showed elongated spindle cells, similar to their in vitro counterparts, which immunostained for MSC markers. Generation of new elastic fibers was evident by both special stains and antibodies to human elastin. The application of cultured cells was safe, without treatment-related adverse events. A strong direct correlation was found between the number of cells applied (greater than 1 x 10(6) cells per cm2 of wound area) and the subsequent decrease in chronic wound size (p = 0.0058). Topical application of autologous MSC also stimulated closure of full-thickness wounds in diabetic mice (db/db). Tracking of green fluorescent protein (GFP)+ MSC in mouse wounds showed GFP+ blood vessels, suggesting that the applied cells may persist as well as act to stimulate the wound repair process. These findings indicate that autologous bone marrow-derived MSC can be safely and effectively delivered to wounds using a fibrin spray system.     

The interplay between extracellular matrix and progenitor/stem cells during wound healing: Opportunities and future directions

Skin wound healing, a dynamic physiological process, progresses through coordinated overlapping phases to restore skin integrity. In some pathological conditions such as diabetes, wounds become chronic and hard-to-heal resulting in substantial morbidity and healthcare costs. Despite much advancement in understanding mechanisms of wound healing, chronic and intractable wounds are still a considerable challenge to nations’ health care systems. Extracellular matrix (ECM) components play pivotal roles in all phases of wound healing. Therefore, a better understanding of their roles during wound healing can help improve wound care approaches. The ECM provides a 3D structure and forms the stem cell niche to support stem cell adhesion and survival and to regulate stem cell behavior and fate. Also, this dynamic structure reserves growth factors, regulates their bioavailability and provides biological signals. In various diseases, the composition and stiffness of the ECM is altered, which as a result, disrupts bidirectional cell-ECM interactions and tissue regeneration. Hence, due to the impact of ECM changes on stem cell fate during wound healing and the possibility of exploring new strategies to treat chronic wounds through manipulation of these interactions, in this review, we will discuss the importance/impact of ECM in the regulation of stem cell function and behavior to find ideal wound repair and regeneration strategies. We will also shed light on the necessity of using ECM in future wound therapy and highlight the potential roles of various biomimetic and ECM-based scaffolds as functional ECM preparations to mimic the native stem cell niche.     

High-efficiency matrix modulus-induced cardiac differentiation of human mesenchymal stem cells inside a thermosensitive hydrogel

Mesenchymal stem cells (MSCs) experience an extremely low rate of cardiac differentiation after transplantation into infarcted hearts, in part due to the inability of stiff scar tissue to support differentiation. We hypothesized that delivering MSCs in a hydrogel with a modulus matched to that of native heart tissue should stimulate MSC differentiation into cardiac cells. We have developed a thermosensitive and injectable hydrogel suitable for the delivery of cells into the heart, and found that the appropriate gel modulus can differentiate MSCs into cardiac cells with high efficiency. The hydrogel was based on N-isopropylacrylamide, N-acryloxysuccinimide, acrylic acid and poly(trimethylene carbonate)-hydroxyethyl methacrylate. The hydrogel solution can be readily injected through needles commonly used for heart injection, and is capable of gelling within 7s at 37°C. The formed gels were highly flexible, with breaking strains (>300%) higher than that of native heart tissue and moduli within the range of native heart tissue (1-140kPa). Controlling the concentration of the hydrogel solution resulted in hydrogels with three different moduli: 16, 45 and 65kPa. The moduli were decoupled from the gel water content and oxygen diffusion, parameters that can also influence cell differentiation. MSCs survived in the hydrogels throughout the entire culture period, and it was observed that gel stiffness did not affect cell survival. After 14days of culture, more than 76% of MSCs had differentiated into cardiac cells in the 45 and 65kPa gels, as confirmed by the expression of cardiac markers at both the gene and protein levels. MSCs in the hydrogel with the 65kPa modulus had the highest differentiation efficiency. The differentiated cells also developed calcium channels that imparted an electrophysiological property, and gap junctions for cell-cell communication. The efficiency of differentiation reported in this study was much higher than for the differentiation approaches described in the literature, such as chemical induction and co-culture of MSCs and cardiomyocytes. These results indicate that the novel hydrogel holds great promise for delivering MSCs into an infarcted heart for the generation of new heart tissue.     

Fibronectin-hyaluronic acid composite hydrogels for three-dimensional endothelial cell culture

Biomaterials that actively promote both wound healing and angiogenesis are of critical importance for many biomedical applications, including tissue engineering. In particular, hyaluronic acid (HA) is an important player that has multiple roles throughout the angiogenic process in the body. Previously, our laboratory has developed photocrosslinkable HA-based scaffolds that promote angiogenesis when implanted in vivo. This paper reports the incorporation of a photocrosslinkable fibronectin (FN) conjugate into three-dimensional (3-D) HA hydrogel networks to enhance endothelial cell adhesion and angiogenesis. The results demonstrate significantly better retention of FN that was photocrosslinked within HA hydrogels compared to FN that was physically adsorbed within HA hydrogels. Increased viability of endothelial cells cultured in 3-D HA hydrogels with photoimmobilized FN, compared to adsorbed FN, was also observed. Endothelial cells were cultured within hydrogels for up to 6 days, a period over which cell proliferation, migration and an angiogenic phenotype were influenced by varying the concentration of incorporated FN. The results demonstrate the potential of these composite hydrogels as biomaterial scaffolds capable of promoting wound healing and angiogenesis.     

In situ forming hydrogels with long-lasting miR-21 enhances the therapeutic potential of MSC by sustaining stimulation of target gene

Regulating stem cells by microRNA (miRNA) is promising in regenerative medicine. However, non-viral transfection is usually transient while stably lentiviral transfection is accompanied with oncogenic risk. In the study, we explored the feasibility to retain the microRNAs within biopolymer hydrogels for their long-lasting working and sustaining stimulation of target gene. miRNA-21 (MiR-21), a reported microRNA enhancing the therapeutic potential of mesenchymal stem cells (MSCs) was used. We demonstrated that miR-21 could be efficiently retained within collagen hydrogel after forming complex with cationic polymer polyethylenimine (PEI). Due to the electronic interaction with positively charged PEI, the release of miR-21 was largely prevented during 2 week incubation (<20%), while free miR-21 encapsulated in hydrogels was largely released (>50%). When MSCs were cultivated in the PEI/miR-21-incorporated hydrogels, the sustained activation of targeted gene HIF-1α was observed, resulting in the sustaining up-regulation of several downstream therapeutic cytokines. Then, the hydrogels encapsulating miR-21/PEI were coated onto tissue plate for MSC cultivation, which further confirmed the long-lasting retention and efficacy of miR-21 on the plate surface. In addition, under H₂O₂-simulated stress condition, we also demonstrated that the anti-apoptotic capacity of MSCs was significantly improved when growing on miR-21-retained hydrogels. Our study provided a safe and promising method for long-lasting stem cell regulation with miRNAs.     

DNA delivery from matrix metalloproteinase degradable poly(ethylene glycol) hydrogels to mouse cloned mesenchymal stem cells

The ability to genetically modify mesenchymal stem cells (MSCs) seeded inside synthetic hydrogel scaffolds would offer an alternative approach to guide MSC differentiation and to study molecular pathways in three dimensions than protein delivery. In this report, we explored gene transfer to infiltrating MSCs into matrix metalloproteinase (MMP) degradable hydrogels that were loaded with DNA/poly(ethylene imine) (PEI) polyplexes. DNA/PEI polyplexes were encapsulated inside poly(ethylene glycol) (PEG) hydrogels crosslinked with MMP-degradable peptides via Michael addition chemistry. A large fraction of encapsulated polyplexes remained active after encapsulation (65%) and the mechanical properties of the hydrogels were unchanged by the encapsulation of the polyplexes. Cells were seeded inside the hydrogel scaffolds using two different approaches: clustered and homogeneous. The viability of MSCs was similar in hydrogels with and without polyplexes. Transgene expression was characterized with time using a secreted reporter gene and showed different profiles for clustered and homogeneously seeded cells. Clustered cells resulted in cumulative transgene expression that increased through the 21-day incubation, while homogeneously seeded cells resulted in cumulative transgene expression that plateaued after 7 days of culture. The use of hydrogel scaffolds that allow cellular infiltration to deliver DNA may result in long lasting signals in vivo, which are essential for the regeneration of functional tissues.