Combination of stromal cell‐derived factor‐1 and collagen–glycosaminoglycan scaffold delays contraction and accelerates reepithelialization of dermal wounds in wild‐type mice

While dermal substitutes can mitigate scarring and wound contraction, a significant drawback of current dermal replacement technologies is the apparent delay in vascular ingrowth compared with conventional skin grafts. Herein, we examined the effect of the chemokine stromal cell‐derived factor‐1 (SDF‐1) on the performance of a porous collagen–glycosaminoglycan dermal analog in excisional wounds in mice. C57BL/6 mice with 1 cm × 1 cm dorsal full‐thickness wounds were covered with a collagen–glycosaminoglycan scaffold, followed by four daily topical applications of 1 μg SDF‐1 or phosphate‐buffered saline vehicle. Some animals were also pretreated with five daily doses of 300 mg/kg granulocyte colony‐stimulating factor. Animals treated with SDF‐1 and no granulocyte colony‐stimulating factor reepithelialized 36% faster than vehicle controls (16 vs. 25 days), and exhibited less wound contraction on postwounding day 18 (～35% greater wound area) plus three‐fold longer neoepidermis formed than controls. Conversely, granulocyte colony‐stimulating factor promoted contraction and no epidermal regeneration. Early (postwounding Day 3) inflammatory cell infiltration in the SDF‐1‐treated group was 86% less, while the fraction of proliferating cells (positive Ki67 staining) was 32% more, when compared with controls. These results suggest that SDF‐1 simultaneously delays contraction and promotes reepithelialization and may improve the wound‐healing performance of skin substitutes.     

Alternatively activated macrophages derived from THP‐1 cells promote the fibrogenic activities of human dermal fibroblasts

Macrophages play a key role in the wound healing process and can be divided into classically activated macrophages (M1) and alternatively activated macrophages (M2). Fibroblasts maintain the physical integrity of connective tissue, participate in wound closure as well as produce and remodel extracellular matrix. Macrophages have a close relationship with fibroblasts by increasing the production of matrix metalloproteinase‐1 (MMP‐1) for faster wound closure and remodeling and myofibroblast differentiation from fibroblasts. In this study, resting state (M0), M1 and M2 macrophages differentiated from the human monocytic THP‐1 cell line were used to co‐culture with human dermal fibroblasts (HDF) for 48, 96 and 144 hours to investigate the effect of macrophages subsets on the fibrogenic activity of fibroblasts. The differentiation and polarization from THP‐1 cells to M0, M1 and M2 macrophages were characterized by flow cytometry and cell cycle analysis. Cell sorting was performed to purify M0 and M2 macrophages. Cell proliferation, collagen synthesis, myofibroblast formation, gene expression of anti‐fibrotic and pro‐fibrotic factors, MMP‐1 activity, and cytokine concentration were investigated. Results showed differentiation of M0 and polarization of M1 and M2 macrophages. M2 macrophages promoted the fibrogenic activities of co‐cultured HDF by facilitating cell proliferation, increasing the collagen content, alpha‐smooth muscle actin expressed cells, expression of the pro‐fibrotic genes and concentration of M2 macrophage related factors, as well as decreasing the expression of the anti‐fibrotic genes and MMP‐1 activity. These findings reinforce the pro‐fibrotic role of M2 macrophages, suggesting therapeutic strategies in fibrotic diseases should target M2 macrophages in the future.     

Hypoxia pretreatment of bone marrow—derived mesenchymal stem cells seeded in a collagen‐chitosan sponge scaffold promotes skin wound healing in diabetic rats with hindlimb ischemia

Bone marrow—derived mesenchymal stem cells (BM‐MSCs) have properties that make them promising for the treatment of chronic nonhealing wounds. The major challenge is ensuring an efficient, safe, and painless delivery of BM‐MSCs. Tissue‐engineered skin substitutes have considerable benefits in skin damage resulting from chronic nonhealing wounds. Here, we have constructed a three‐dimensional biomimetic scaffold known as collagen‐chitosan sponge scaffolds (CCSS) using the cross‐linking and freeze‐drying method. Scanning electron microscopy images showed that CCSS had an interconnected network pore configuration about 100 μm and exhibited a suitable swelling ratio for maintaining morphological stability and appropriate biodegradability to improve biostability using swelling and degradation assays. Furthermore, BM‐MSCs were seeded in CCSS using the two‐step seeding method to construct tissue‐engineered skin substitutes. In addition, in this three‐dimensional biomimetic CCSS, BM‐MSCs secreted their own collagen and maintain favorable survival ability and viability. Importantly, BM‐MSCs exhibited a significant upregulated expression of proangiogenesis factors, including HIF‐1α, VEGF, and PDGF following hypoxia pretreatment. In vivo, hypoxia pretreatment of the skin substitute observably accelerated wound closure via the reduction of inflammation and enhanced angiogenesis in diabetic rats with hindlimb ischemia. Thus, hypoxia pretreatment of the skin substitutes can serve as ideal bioengineering skin substitutes to promote optimal diabetic skin wound healing.     

Cross‐linking affects cellular condensation and chondrogenesis in type II collagen‐GAG scaffolds seeded with bone marrow‐derived mesenchymal stem cells

The formation of cartilaginous tissue by chondroprogenitor cells, whether in vivo or in vitro, appears to require a critical initial stage of “condensation” in which intercellular space is reduced through an aggregation of cells, leading to development of cell‐to‐cell junctions followed by chondrocytic differentiation. The objective of this study was to investigate the association of aggregation (condensation) of mesenchymal stem cell (MSCs) and chondrogenesis in vitro. Previous work with chondrocytes indicated that the cross‐link density and related cell‐mediated contraction of collagen scaffolds significantly affects cartilaginous tissue formation within the cell‐seeded construct. Based on this finding, we hypothesized that the cell‐aggregating effect of the contraction of MSC‐seeded collagen scaffolds of lower cross‐link density favors chondrogenesis; scaffolds of higher cross‐link density, which resist cell‐mediated contraction, would demonstrate a lower cell number density (i.e., subcritical packing density) and less cartilage formation. Type II collagen–GAG scaffolds, chemically cross‐linked to achieve a range of cross‐link densities, were seeded with caprine MSCs and cultured for 4 weeks. Constructs with low cross‐link densities experienced cell‐mediated contraction, increased cell number densities, and a greater degree of chondrogenesis (indicated by the chondrocytic morphology of cells, and synthesis of GAG and type II collagen) compared to more highly cross‐linked scaffolds that resisted cellular contraction. These results provide a foundation for further investigation of the mechanisms by which condensation of mesenchymal cells induces chondrogenesis in this in vitro model, and may inform cross‐linking protocols for collagen scaffolds for use in cartilage tissue engineering. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1184–1192, 2010     

Deep dermal fibroblast profibrotic characteristics are enhanced by bone marrow–derived mesenchymal stem cells

Hypertrophic scars are a significant fibroproliferative disorder complicating deep injuries to the skin. We hypothesize that activated deep dermal fibroblasts are subject to regulation by bone marrow–derived mesenchymal stem cells (BM‐MSCs), which leads to the development of excessive fibrosis following deep dermal injury. We found that the expression of fibrotic factors was higher in deep burn wounds compared with superficial burn wounds collected from burn patients with varying depth of skin injury. We characterized deep and superficial dermal fibroblasts, which were cultured from the deep and superficial dermal layers of normal uninjured skin obtained from abdominoplasty patients, and examined the paracrine effects of BM‐MSCs on the fibrotic activities of the cells. In vitro, deep dermal fibroblasts were found higher in the messenger RNA (mRNA) levels of type 1 collagen, alpha smooth muscle actin, transforming growth factor beta, stromal cell–derived factor 1, and tissue inhibitor of metalloproteinase 1, an inhibitor of collagenase (matrix metalloproteinase 1). As well, deep dermal fibroblasts had low matrix metalloproteinase 1 mRNA, produced more collagen, and contracted collagen lattices significantly greater than superficial fibroblasts. By co‐culturing layered fibroblasts with BM‐MSCs in a transwell insert system, BM‐MSCs enhanced the fibrotic behavior of deep dermal fibroblasts, which suggests a possible involvement of BM‐MSCs in the pathogenesis of hypertrophic scarring.     

Adipose‐derived stem cells and keratinocytes in a chronic wound cell culture model: the role of hydroxyectoine

Chronic wounds represent a major socio‐economic problem in developed countries today. Wound healing is a complex biological process. It requires a well‐orchestrated interaction of mediators, resident cells and infiltrating cells. In this context, mesenchymal stem cells and keratinocytes play a crucial role in tissue regeneration. In chronic wounds these processes are disturbed and cell viability is reduced. Hydroxyectoine (HyEc) is a membrane protecting osmolyte with protein and macromolecule stabilising properties. Adipose‐derived stem cells (ASC) and keratinocytes were cultured with chronic wound fluid (CWF) and treated with HyEc. Proliferation was investigated using MTT test and migration was examined with transwell‐migration assay and scratch assay. Gene expression changes of basic fibroblast growth factor (b‐FGF), vascular endothelial growth factor (VEGF), matrix metalloproteinases‐2 (MMP‐2) and MMP‐9 were analysed by quantitative real‐time polymerase chain reaction (qRT‐PCR). CWF significantly inhibited proliferation and migration of keratinocytes. Addition of HyEc did not affect these results. Proliferation capacity of ASC was not influenced by CWF whereas migration was significantly enhanced. HyEc significantly reduced ASC migration. Expression of b‐FGF, VEGF, MMP‐2 and MMP‐9 in ASC, and b‐FGF, VEGF and MMP‐9 in keratinocytes was strongly induced by chronic wound fluid. HyEc enhanced CWF induced gene expression of VEGF in ASC and MMP‐9 in keratinocytes. CWF negatively impaired keratinocyte function, which was not influenced by HyEc. ASC migration was stimulated by CWF, whereas HyEc significantly inhibited migration of ASC. CWF induced gene expression of VEGF in ASC and MMP‐9 in keratinocytes was enhanced by HyEc, which might partly be explained by an RNA stabilising effect of HyEc.     

Influence of age‐related degeneration on regenerative potential of human nucleus pulposus cells

Nucleus pulposus (NP) cells, sourced from herniation surgeries, may be used as a cell‐based therapy for intervertebral disc (IVD) degeneration. But, both the regenerative potential of these degenerative adult NP cells and how to stimulate optimum matrix synthesis is not yet clear. The purpose of the current study was to understand the different phenotypic behaviors between degenerative adult NP cells and normal adolescent NP cells. Degenerative adult NP cells produced a significantly higher amount of proteoglycans and collagens than adolescent cells. Insulin‐like growth factor‐1 was the only anabolic cytokine with increased endogenous expression in degenerative adult NP cells. TGF‐β1 treatment of degenerative NP cells promoted matrix synthesis but stimulated too much type I collagen and suppressed type II collagen and aggrecan. Adult degenerative NP cells possess upregulated regenerative potential, but stimulation in addition to TGF‐β1 is needed to enhance matrix productivity and optimize the collagen expression profile. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:379–383, 2010     

A novel injectable scaffold for cartilage tissue engineering using adipose‐derived adult stem cells

Articular cartilage has a limited self‐regenerative capacity. Thus, treatment of cartilage lesions is a major challenge. Tissue engineering using a variety of biomaterials is a promising solution to the problem of cartilage damage. In this in vitro study, we investigated the effect of the presence of cartilage‐tissue chondroitin‐sulfate (CS) in a fibrin scaffold on the differentiation of adipose‐derived adult stem cells (ADAS cells) into chondrocytes. Isolated rabbit ADAS cells were cultured in fibrin matrices with and without CS for up to 14 days. ADAS cells differentiated into chondrocytes in both matrices, but cell proliferation, glycoaminoglycans content, and type II collagen expression were significantly higher in the fibrin–CS matrices than those in the fibrin matrices alone. Histological examination and scanning electronic microscopy revealed the fibrin–CS matrices exceeded in inducing differentiation of ADAS cells into chondrocytes in terms of tissue morphological characteristics. We concluded that the fibrin–CS matrices mimicking native cartilage extracellular matrix could act as a three‐dimensional scaffold for cartilage tissue engineering and have the potential for promoting ADAS cells differentiation into chondrocytes. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:27–33, 2008     

Adipose‐derived stromal cell therapy combined with a short course nonmyeloablative conditioning promotes long‐term graft tolerance in vascularized composite allotransplantation

The risks of chronic immunosuppression limit the utility of vascularized composite allotransplantation (VCA) as a reconstructive option in complex tissue defects. We evaluated a novel, clinically translatable, radiation‐free conditioning protocol that combines anti‐lymphocyte serum (ALS), tacrolimus, and cytotoxic T‐lymphocyte‐associated protein 4 immunoglobulin (CTLA4‐Ig) with adipose‐derived stromal cells (ASCs) to allow VCA survival without long‐term systemic immunosuppression. Full‐mismatched rat hind‐limb‐transplant recipients received tacrolimus (0.5 mg/kg) for 14 days and were assigned to 4 groups: controls (CTRL) received no conditioning; ASC‐group received CTLA4‐Ig (10 mg/kg body weight i.p. postoperative day [POD] 2, 4, 7) and donor ASCs (1 × 10⁶ iv, POD 2, 4, 7, 15, 28); the ASC‐cyclophosphamide (CYP)‐group received CTLA4‐Ig, ASC plus cyclophosphamide (50 mg/kg ip, POD 3); the ASC‐ALS‐group received CTLA4‐Ig, ASCs plus ALS (500 µL ip, POD 1, 5). Banff grade III or 120 days were endpoints. ASCs suppressed alloresponse in vitro. Median rejection‐free VCA survival was 28 days in CTRL (n = 7), 34 in ASC (n = 6), and 27.5 in ASC‐CYP (n = 4). In contrast, ASC‐ALS achieved significantly longer, rejection‐free VCA survival in 6/7 animals (86%), with persistent mixed donor‐cell chimerism, and elevated systemic and allograft skin T_regs, with no signs of acute cellular rejection. Taken together, a regimen comprised of short‐course tacrolimus, repeated CTLA4‐Ig and ASC administration, combined with ALS, promotes long‐term VCA survival without chronic immunosuppression.     

The regenerative role of adipose‐derived stem cells (ADSC) in plastic and reconstructive surgery

The potential use of stem cell‐based therapies for the repair and regeneration of various tissues and organs offers a paradigm shift in plastic and reconstructive surgery. The use of either embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC) in clinical situations is limited because of regulations and ethical considerations even though these cells are theoretically highly beneficial. Adult mesenchymal stem cells appear to be an ideal stem cell population for practical regenerative medicine. Among these cells, adipose‐derived stem cells (ADSC) have the potential to differentiate the mesenchymal, ectodermal and endodermal lineages and are easy to harvest. Additionally, adipose tissue yields a high number of ADSC per volume of tissue. Based on this background knowledge, the purpose of this review is to summarise and describe the proliferation and differentiation capacities of ADSC together with current preclinical data regarding the use of ADSC as regenerative tools in plastic and reconstructive surgery.     

Mesenchymal stem cells and platelet lysate in fibrin or collagen scaffold promote non‐cemented hip prosthesis integration

The objective of this study was to evaluate whether mesenchymal stem cells (MSC) and platelet lysate (PL) seeded in a fibrin or collagen scaffold could improve the new bone (NB) formation around an uncemented hip prosthesis stem in a sheep model. In vitro expanded MSC were suspended in PL and either mixed with collagen or fibrin gel as delivery vehicle. The cell–gel composites were inserted inside the femoral canal, then the prosthesis was press‐fit inserted inside the femur. Identical procedures were performed in a control group, but only the prosthesis was implanted. Histomorphometrical analysis performed 4 months after surgery indicated that the newly formed bone inside the medullary canal, between the inner cortex and the prosthetic stem, was significantly higher in the MSC–PL–collagen group (mean 18.7 ± 4.5%) and in the MSC–PL–fibrin group (mean 18.8 ± 15.2%) when compared to the control group (mean 4.6 ± 2.0%). There was a significantly higher bone–prosthesis contact in the MSC–PL–collagen group (mean 2.7 ± 2.6%) and in the MSC–PL–fibrin group (mean 2.3 ± 3.1%) compared to the control group (mean 0.2 ± 0.1%). The results indicate that MSC and PL in a fibrin or collagen scaffold can promote NB formation around an uncemented hip prosthesis stem. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:961–968     

Examining the potential for porcine‐derived islet cells to harbour viral pathogens

With an onus on safety in the potential use of porcine islet cells as a treatment for diabetes, the use of animals lacking exogenous pathogens is clearly important and multilevel screening strategies have been presented on testing animals and the product. In this study, we wished to investigate whether islet cells indeed harboured the same viral pathogens of concern in the source animal. PMBC and islet cells from both adult and neonatal source animals were directly compared and tested for PCMV, PLHV, PCV2, PPV and HEV using both molecular and serological assays. Adult PBMC were found positive for all viruses with the exception of PCV2 and HEV. Neonatal PBMC were only found positive for PCMV and HEV. All animals were found negative for HEV antibodies. Interestingly, islet cells were negative for all viruses tested regardless of status in the animal‐derived PBMC. Given that other laboratories have demonstrated the lack of virus detection during the culture of islets, this study also demonstrates that the hygiene status of the herd may not reflect the status of the product. This is important for establishing guidelines for any risk evaluation and mitigation process utilised during product manufacture.     

Neovascular potential of adipose‐derived stromal cells (ASCs) from diabetic patients

We explored the vascular biology of adipose‐derived stromal cells (ASCs) from diabetic patients and applied these cells to a murine ischemic flap model to assess the comparative angiogenic potentials between normal and diabetic human ASCs. ASCs were obtained from diabetic patients (n = 5) and controls (n = 5). Secretion and expression of angiogenic cytokines were measured under normoxic and hypoxic condition in vitro. Conditioned media harvested from ASC cultures were assessed for their ability to stimulate human umbilical vein endothelial cell proliferation and tubulization. The control and diabetic ASCs were injected into the murine ischemic flaps, and the surviving area was measured. Diabetic adipose‐derived stromal cells showed a lower level of vascular endothelial growth factor expression and cell proliferation rates than the control cells (p < 0.05). However, vascular endothelial growth factor, hepatocyte growth factor secretion, tubulogenesis, and cell proliferation in diabetic conditioned media were increased in response to hypoxic stimuli (p < 0.05), and it was similar to those of control cells. In an animal study, diabetic and normal ASCs significantly increased flap survival (p < 0.05); however, the functional difference was not found between the two groups. Diabetic ASCs were impaired in their ability to produce vascular endothelial growth factors and to induce cellular proliferation under hypoxic conditions. However, diabetic ASCs showed similar flap salvaging effect compared with controls. These findings may be important in the context of future study of autologous cell‐based therapy in diabetic patients.