H3K27Me3 abundance increases fibrogenesis during endothelial-to-mesenchymal transition via the silencing of microRNA-29c.

Objective: Endothelial-to-mesenchymal transition (EndMT) is a transdifferentiation process in which endothelial cells (ECs) adopt a mesenchymal-like phenotype. Over the past few years, it became clear that EndMT can contribute to several cardiovascular pathologies. However, the molecular pathways underlying the development of EndMT remain incompletely understood. Since the epigenetic enzyme Enhancer of Zeste Homolog 2 (EZH2) and its concomitant mark H3K27Me3 have been shown to be elevated in many cardiovascular diseases that associate with EndMT, we hypothesized that H3K27Me3 is a determinant for the susceptibility of EndMT. Methods: To study the association between H3K27Me3 and EndMT, a knockdown model of EZH2 in human endothelial cells (HUVEC) was utilized to reduce H3K27Me3 abundance, followed by induction of EndMT using TGFß1. The expression of molecular markers of EndMT and fibrogenesis were analysed. Results: In cultured HUVECs, a reduction of H3K27Me3 abundance facilitates EndMT but mitigates fibrogenesis as shown by a decreased expression of collagen I and III. In HUVEC, H3K27Me3 abundance directly affects the expression of miR29c, a collagen-targeting miRNA. Additionally, knockdown of miR-29c in HUVEC with low H3K27Me3 abundance partly restored the expression of collagen I and III. Expectedly, in rats with perivascular fibrosis an increased abundance of H3K27Me3 associated with a decreased expression of miR-29c. Conclusion: our data shows that endothelial fibrogenesis underlies an epigenetic regulatory pathway and we demonstrate that a decreased abundance of H3K27Me3 in ECs blunts fibrogenesis in part in a miR-29c dependent manner. Therefore, a reduction of H3K27Me3 could serve as a novel therapeutical strategy to mitigate fibrogenesis and may prove to be beneficial in fibrogenic diseases including atherosclerosis, cardiac fibrosis, and PAH.

Fibroblast alignment and matrix remodeling induced by a stiffness gradient in a skin-derived extracellular matrix hydrogel.

Large skin injuries heal as scars. Stiffness gradually increases from normal skin to scar tissue (20x higher), due to excessive deposition and crosslinking of extracellular matrix (ECM) mostly produced by (myo)fibroblasts. Using a custom mold, skin-derived ECM hydrogels (dECM) were UV crosslinked after diffusion of ruthenium (Ru) to produce a Ru-dECM gradient hydrogel. The Ru diffusion gradient equates to a stiffness gradient and models physiology of the scarred skin. Crosslinking in Ru-dECM hydrogels results in a 23-fold increase in stiffness from a stiffness similar to that of normal skin. Collagen fiber density increases in a stiffness-dependent fashion while stress relaxation also alters, with one additional Maxwell element necessary for characterizing Ru-dECM. Alignment of fibroblasts encapsulated in hydrogels suggests that the stiffness gradient directs fibroblasts to orientate at ∼45 ° in regions below 120 kPa. In areas above 120 kPa, fibroblasts decrease the stiffness prior to adjusting their orientation. Furthermore, fibroblasts remodel their surrounding ECM in a gradient-dependent fashion, with rearrangement of cell-surrounding ECM in high-stiffness areas, and formation of interlaced collagen bundles in low-stiffness areas. Overall, this study shows that fibroblasts remodel their local environment to generate an optimal ECM mechanical and topographical environment. STATEMENT OF SIGNIFICANCE: This study developed a versatile in vitro model with a gradient stiffness using skin-derived ECM hydrogel with unchanged biochemical environment. Using Ruthenium crosslinking, a 20-fold stiffness increase was achieved as observed in fibrotic skin. The interaction between fibroblasts and matrix depends on changes in the matrix stiffness. The stiffness gradient directed the alignment of fibroblasts with ∼45° in regions with≤ 120 kPa. The cells in regions with the higher stiffness decreased stiffness first and then oriented themselves. Furthermore, fibroblasts remodeled surrounding ECM and regulated its mechanics in a gradient-dependent fashion to reach an optimal condition. Our study highlights the dynamic interplay between cells and surrounding matrix, shedding light on potential mechanisms and strategies to target scar formation and remodeling.

Physical Properties and Biochemical Composition of Extracellular Matrix-Derived Hydrogels Dictate Vascularization Potential in an Organ-Dependent Fashion.

The inherent extracellular matrix (ECM) originating from a specific tissue impacts the process of vascularization, specifically vascular network formation (VNF) orchestrated by endothelial cells (ECs). The specific contribution toward these processes of ECM from highly disparate organs such as the skin and lungs remains a relatively unexplored area. In this study, we compared VNF and ECM remodeling mediated by microvascular ECs within gel, lung, and combinations thereof (hybrid) ECM hydrogels. Irrespective of the EC source, the skin-derived ECM hydrogel exhibited a higher propensity to drive and support VNF compared to both lung and hybrid ECM hydrogels. There were distinct disparities in the physical properties of the three types of hydrogels, including viscoelastic properties and complex architectural configurations, including fiber diameter, pore area, and numbers among the fibers. The hybrid ECM hydrogel properties were unique and not the sum of the component ECM parts. Furthermore, cellular ECM remodeling responses varied with skin ECM hydrogels promoting matrix metalloproteinase 1 (MMP1) secretion, while hybrid ECM hydrogels exhibited increased MMP9, fibronectin, and collagen IV deposition. Principal component analysis (PCA) indicated that the influence of a gel's mechanical properties on VNF was stronger than the biochemical composition. These data indicate that the organ-specific properties of an ECM dictate its capacity to support VNF, while intriguingly showing that ECs respond to more than just the biochemical constituents of an ECM. The study suggests potential applications in regenerative medicine by strategically selecting ECM origin or combinations to manipulate vascularization, offering promising prospects for enhancing wound healing through pro-regenerative interventions.

Fibroblasts alter the physical properties of dermal ECM-derived hydrogels to create a pro-angiogenic microenvironment.

This study aimed to investigate the impact of fibroblasts (MRC-5) on the extracellular matrix (ECM) microenvironment of endothelial cells (ECs) during the vascularization of skin-derived ECM hydrogel in vitro. Two types of ECs were studied: human dermal microvascular endothelial cells (HMEC) and human pulmonary microvascular endothelial cells (HPMEC). Results showed that the presence of MRC-5 fibroblasts increased the stiffness of the hydrogel and led to larger fiber diameters and increased porosity. Extensive collagen fiber remodeling occurred in the ECM hydrogel with MRC-5 fibroblasts. Additionally, higher levels of fibulin-1 and fibronectin were deposited in the hydrogel when co-cultured with MRC-5 fibroblasts. These findings suggest that MRC-5 fibroblasts play a role in modifying the ECM microenvironment, promoting vascularization through dynamic ECM remodeling.

Limited Efficacy of Adipose Stromal Cell Secretome-Loaded Skin-Derived Hydrogels to Augment Skin Flap Regeneration in Rats.

Insufficient vascularization is a recurring cause of impaired pedicled skin flap healing. The administration of adipose tissue-derived stromal cells' (ASCs') secretome is a novel approach to augment vascularization. Yet, the secretome comprised of soluble factors that require a sustained-release vehicle to increase residence time. We hypothesized that administration of a hydrogel derived from decellularized extracellular matrix (ECM) of porcine skin with bound trophic factors from ASCs enhances skin flap viability and wound repair in a rat model. Porcine skin was decellularized and pepsin-digested to form a hydrogel at 37°C. Conditioned medium (CMe) of human ASC was collected, concentrated 20-fold, and mixed with the hydrogel. Sixty Wistar rats were included. A dorsal skin flap (caudal based) of 3 × 10 cm was elevated for topical application of DMEM (group I), a prehydrogel with or without ASC CMe (groups II and III), or ASC CMe (group IV). After 7, 14, and 28 days, perfusion was measured, and skin flaps were harvested for wound healing assessment and immunohistochemical analysis. Decellularized skin ECM hydrogel contained negligible amounts of DNA (11.6 ± 0.6 ng/mg), was noncytotoxic and well tolerated by rats. Irrespective of ASC secretome, ECM hydrogel application resulted macroscopically and microscopically in similar dermal wound healing in terms of proliferation, immune response, and matrix remodeling as the control group. However, ASC CMe alone increased vessel density after 7 days. Porcine skin-derived ECM hydrogels loaded with ASC secretome are noncytotoxic but demand optimization to significantly augment wound healing of skin flaps.

Autologous Lipofilling Improves Clinical Outcome in Patients With Symptomatic Dermal Scars Through Induction of a Pro-Regenerative Immune Response.

BACKGROUND: Autologous lipofilling is an emerging procedure to treat and possibly reverse dermal scars and to reduce scar-related pain, but its efficacy and mechanisms are poorly understood. OBJECTIVES: The aim of this study was to test the hypothesis that repeated lipografts reverse dermal scars by reinitiation of wound healing. METHODS: In a prospective, non-placebo-controlled clinical study, 27 adult patients with symptomatic scars were given 2 lipofilling treatments at 3-month intervals. As primary outcome, clinical effects were measured with the Patient and Observer Scar Assessment Scale (POSAS). Scar biopsies were taken before and after treatments to assess scar remodeling at a cellular level. RESULTS: Twenty patients completed the study. Patients' scars improved after lipofilling. The total POSAS scores (combined patient and observer scores) decreased from 73.2  [14.7] points (mean [standard deviation]) pretreatment to 46.1 [14.0] and 32.3 [13.2] points after the first and second lipofilling treatment, respectively. Patient POSAS scores decreased from 37.3 [8.8] points to 27.2 [11.3] and 21.1 [11.4] points, whereas observer POSAS scores decreased from 35.9 [9.5] points to 18.9 [6.0] and 11.3 [4.5] points after the first and second treatment, respectively. After each lipofilling treatment, T lymphocytes, mast cells, and M2 macrophages had invaded scar tissue and were associated with increased vascularization. In addition, the scar-associated epidermis showed an increase in epidermal cell proliferation to levels similar to that normal in skin. Moreover, lipofilling treatment caused normalization of the extracellular matrix organization towards that of normal skin. CONCLUSIONS: Autologous lipofilling improves the clinical outcome of dermal scars through the induction of a pro-regenerative immune response, increased vascularization, and epidermal proliferation and remodeling of scar tissue extracellular matrix.

Tissue Stromal Vascular Fraction Improves Early Scar Healing: A Prospective Randomized Multicenter Clinical Trial.

BACKGROUND: Wound healing and scar formation depends on a plethora of factors. Given the impact of abnormal scar formation, interventions aimed to improve scar formation would be most advantageous. The tissue stromal vascular fraction (tSVF) of adipose tissue is composed of a heterogenous mixture of cells embedded in extracellular matrix. It contains growth factors and cytokines involved in wound-healing processes, eg, parenchymal proliferation, inflammation, angiogenesis, and matrix remodeling. OBJECTIVES: The aim of this study was to investigate the hypothesis that tSVF reduces postsurgical scar formation. METHODS: This prospective, double-blind, placebo-controlled, randomized trial was conducted between 2016 and 2020. Forty mammoplasty patients were enrolled and followed for 1 year. At the end of the mammoplasty procedure, all patients received tSVF in the lateral 5 cm of the horizontal scar of 1 breast and a placebo injection in the contralateral breast to serve as an intrapatient control. Primary outcome was scar quality measure by the Patient and Observer Scar Assessment Scale (POSAS). Secondary outcomes were obtained from photographic evaluation and histologic analysis of scar tissue samples. RESULTS: Thirty-four of 40 patients completed follow-up. At 6 months postoperation, injection of tSVF had significantly improved postoperative scar appearance as assessed by the POSAS questionnaire. No difference was observed at 12 months postoperation. No improvement was seen based on the evaluation of photographs and histologic analysis of postoperative scars between both groups. CONCLUSIONS: Injection of tSVF resulted in improved wound healing and reduced scar formation at 6 months postoperation, without any noticeable advantageous effects seen at 12 months.

The Addition of Tissue Stromal Vascular Fraction to Platelet-Rich Plasma Supplemented Lipofilling Does Not Improve Facial Skin Quality: A Prospective Randomized Clinical Trial.

BACKGROUND: Lipofilling has become popular as a treatment to improve aging-related skin characteristics (eg, wrinkles, pigmentation spots, pores, or rosacea). Different additives such as platelet-rich plasma (PRP) or stromal vascular fraction (SVF) have been combined with lipofilling to increase the therapeutic effect of adipose-derived stromal cells (ASCs). OBJECTIVES: The aim of this study was to examine the hypothesis that mechanically isolated SVF augments the therapeutic effect of PRP-supplemented lipofilling to improve facial skin quality. METHODS: This prospective, double-blind, placebo-controlled, randomized trial was conducted between 2016 and 2019. In total, 28 female subjects were enrolled; 25 completed the follow-up. All patients received PRP-supplemented lipofilling with either mechanically isolated SVF or saline. SVF was isolated by fractionation of adipose tissue (tSVF). Results were evaluated by changes in skin elasticity and transepidermal water loss, changes in skin-aging-related features, ie, superficial spots, wrinkles, skin texture, pores, vascularity, and pigmentation, as well as patient satisfaction (FACE-Q), recovery, and number of complications up to 1 year postoperative. RESULTS: The addition of tSVF to PRP-supplemented lipofilling did not improve skin elasticity, transepidermal water loss, or skin-aging-related features. No improvement in patient satisfaction with overall facial appearance or facial skin quality was seen when tSVF was added to PRP-supplemented lipofilling. CONCLUSIONS: In comparison to PRP-supplemented lipofilling, PRP-supplemented lipofilling combined with tSVF does not improve facial skin quality or patient satisfaction in a healthy population. PRP-supplemented lipofilling combined with tSVF can be considered a safe procedure.

Topography-Mediated Myotube and Endothelial Alignment, Differentiation, and Extracellular Matrix Organization for Skeletal Muscle Engineering.

Understanding the response of endothelial cells to aligned myotubes is important to create an appropriate environment for tissue-engineered vascularized skeletal muscle. Part of the native tissue environment is the extracellular matrix (ECM). The ECM is a supportive scaffold for cells and allows cellular processes such as proliferation, differentiation, and migration. Interstitial matrix and basal membrane both comprise proteinaceous and polysaccharide components for strength, architecture, and volume retention. Virtually all cells are anchored to their basal lamina. One of the physical factors that affects cell behavior is topography, which plays an important role on cell alignment. We tested the hypothesis that topography-driven aligned human myotubes promote and support vascular network formation as a prelude to in vitro engineered vascularized skeletal muscle. Therefore, we used a PDMS-based topography substrate to investigate the influence of pre-aligned myotubes on the network formation of microvascular endothelial cells. The aligned myotubes produced a network of collagen fibers and laminin. This network supported early stages of endothelial network formation.

Adipose tissue-derived ECM hydrogels and their use as 3D culture scaffold.

Adipose tissue has the therapeutic capacity in the form of a fat graft, for example, for treatment of irradiation-induced scars and difficult to heal dermal wounds. For large-scale clinical application, an off-the-shelf product is warranted. In recent years, ECM-derived hydrogels are postulated to harbour therapeutic capacity and might even replicate the beneficial effects of adipose tissue. In normal homeostasis, the natural ECM acts as a deposit of growth factors, that releases them over time. In the healing of lesions, this might promote cell accumulation and proliferation which in turn stimulates angiogenesis and repair. The decellularization of tissue and the generation of hydrogels may leave cytotoxic traces. Therefore, our research assessed the cytotoxic effect of human adipose tissue-derived ECM hydrogels on connective tissue cells i.e. fibroblasts. The results showed no cytotoxicity, meaning the hydrogels caused no cell death. Cell migration and survival were observed when cultured in ECM hydrogels and followed for 7 days. Cell survival in the hydrogel was confirmed with CFDA staining and also cells showed the ability to penetrate and migrate throughout the gel. We conclude that ECM hydrogels are promising to use as innovative therapy for wound healing.

Adipose tissue-derived extracellular matrix hydrogels as a release platform for secreted paracrine factors.

Fat grafting is an established clinical intervention to promote tissue repair. The role of the fat's extracellular matrix (ECM) in regeneration is largely neglected. We investigated in vitro the use of human adipose tissue-derived ECM hydrogels as release platform for factors secreted by adipose-derived stromal cells (ASCs). Lipoaspirates from nondiabetic and diabetic donors were decellularized. Finely powdered acellular ECM was evaluated for cell remainders and DNA content. Acellular ECM was digested, and hydrogels were formed at 37°C and their viscoelastic relaxation properties investigated. Release of ASC-released factors from hydrogels was immune assessed, and bio-activity was determined by fibroblast proliferation and migration and endothelial angiogenesis. Acellular ECM contained no detectable cell remainders and negligible DNA contents. Viscoelastic relaxation measurements yielded no data for diabetic-derived hydrogels due to gel instability. Hydrogels released several ASC-released factors concurrently in a sustained fashion. Functionally, released factors stimulated fibroblast proliferation and migration as well as angiogenesis. No difference between nondiabetic and diabetic hydrogels in release of factors was measured. Adipose ECM hydrogels incubated with released factors by ASC are a promising new therapeutic modality to promote several important wound healing-related processes by releasing factors in a controlled way.

The 6-chromanol derivate SUL-109 enables prolonged hypothermic storage of adipose tissue-derived stem cells.

Encouraging advances in cell therapy research with adipose derived stem cells (ASC) require an effective short-term preservation method that provides time for quality control and transport of cells from their manufacturing facility to their clinical destination. Hypothermic storage of cells in their specific growth media offers an alternative and simple preservation method to liquid nitrogen cryopreservation or commercial preservation fluids for short-term storage and transport. However, accumulation of cell damage during hypothermia may result in cell injury and death upon rewarming through the production of excess reactive oxygen species (ROS). Here, the ability of the cell culture medium additive SUL-109, a modified 6-chromanol, to protect ASC from hypothermia and rewarming damage is examined. SUL-109 conveys protective effects against cold-induced damage in ASC as is observed by preservation of cell viability, adhesion properties and growth potential. SUL-109 does not reduce the multilineage differentiation capacity of ASC. SUL-109 conveys its protection against hypothermic damage by the preservation of the mitochondrial membrane potential through the activation of mitochondrial membrane complexes I and IV, and increases maximal oxygen consumption in FCCP uncoupled mitochondria. Consequently, SUL-109 alleviates mitochondrial ROS production and preserves ATP production. In summary, here we describe the generation of a single molecule cell preservation agent that protects ASC from hypothermic damage associated with short-term cell preservation that does not affect the differentiation capacity of ASC.

Endothelial-to-mesenchymal transition contributes to fibro-proliferative vascular disease and is modulated by fluid shear stress.

AIMS: Neointimal hyperplasia is a common feature of fibro-proliferative vascular disease and characterizes initial stages of atherosclerosis. Neointimal lesions mainly comprise smooth muscle-like cells. The presence of these lesions is related to local differences in shear stress. Neointimal cells may arise through migration and proliferation of smooth muscle cells from the media. However, a role for the endothelium as a source of smooth muscle-like cells has largely been disregarded. Here, we investigated the role of endothelial-to-mesenchymal transition (EndMT) in neointimal hyperplasia and atherogenesis, and studied its modulation by shear stress. METHODS AND RESULTS: In human atherosclerotic plaques and porcine aortic tissues, myo-endothelial cells were identified, suggestive for EndMT. Flow disturbance by thoracic-aortic constriction in mice similarly showed the presence of myo-endothelial cells specifically in regions exposed to disturbed flow. While uniform laminar shear stress (LSS) was found to inhibit EndMT, endothelial cells exposed to disturbed flow underwent EndMT, in vitro and in vivo, and showed atherogenic differentiation. Gain- and loss-of-function studies using a constitutive active mutant of MEK5 and short hairpins targeting ERK5 established a pivotal role for ERK5 signalling in the inhibition of EndMT. CONCLUSION: Together, these data suggest that EndMT contributes to neointimal hyperplasia and induces atherogenic differentiation of endothelial cells. Importantly, we uncovered that EndMT is modulated by shear stress in an ERK5-dependent manner. These findings provide new insights in the role of adverse endothelial plasticity in vascular disease and identify a novel atheroprotective mechanism of uniform LSS, namely inhibition of EndMT.

Dependence of neovascularization mechanisms on the molecular microenvironment.

In vivo vascularization of implanted (bio)artificial constructs is essential for their proper function. Vascularization may rely on sprouting angiogenesis, vascular incorporation of bone marrow-derived endothelial cells (BMDECs), or both. Here we investigated the relative contribution of these 2 mechanisms to neovascularization in a mouse model of a foreign body reaction (FBR) to subcutaneously implanted Dacron and in hind limb ischemia (HLI) in relation to the molecular microenvironment at these neovascularization sites. Neovascularization was studied in C57Bl/6 mice reconstituted with enhanced green fluorescent protein (EGFP) transgenic bone marrow. Sprouting angiogenesis, detected using nuclear incorporation of bromodeoxyuridine in endothelial cells was present in both models, whereas vascular incorporation of EGFP(+) BMDECs was restricted to HLI. In HLI, the presence of a pro-angiogenic molecular microenvironment comprising vascular endothelial growth factor, fibroblast growth factor 2, and granulocyte colony-stimulating factor corroborated the importance of these factors for vascular BMDEC incorporation, whereas this microenvironment was absent in FBR. Enhanced mobilization of BMDECs by granulocyte-macrophage colony-stimulating factor administration or by combining HLI and FBR with Dacron did not induce incorporation of BMDECs in FBR neovessels. We conclude that the efficacy of BMDEC-based therapy is not generally warranted, but it depends on the molecular microenvironment in the targeted tissue.

Molecular recognition in poly(epsilon-caprolactone)-based thermoplastic elastomers.

The molecular recognition properties of the hydrogen bonding segments in biodegradable thermoplastic elastomers were explored, aiming at the further functionalization of these potentially interesting biomaterials. A poly(epsilon-caprolactone)-based poly(urea) 2 was synthesized and characterized in terms of mechanical properties, processibility and histocompatibility. Comparison of the data with those obtained from the structurally related poly(urethane urea) 1 revealed that the difference in hard segment structure does not significantly affect the potency for application as a biomaterial. Nevertheless, the small differences in hard block composition had a strong effect on the molecular recognition properties of the hydrogen bonding segments. High selectivity was found for poly(urea) 2 in which bisureidobutylene-functionalized azobenzene dye 3 was selectively incorporated while bisureidopentylene-functionalized azobenzene dye 4 was completely released. In contrast, the incorporation of both dyes in poly(urethane urea) 1 led in both cases to their gradual release in time. Thermal analysis of the polymers in combination with variable temperature infrared experiments indicated that the hard blocks in 1 showed a sharp melting point, whereas those in 2 showed a very broad melting trajectory. This suggests a more precise organization of the hydrogen bonding segments in the hard blocks of poly(urea) 2 compared to poly(urethane urea) 1 and explains the results from the molecular recognition experiments. Preliminary results revealed that a bisureidobutylene-functionalized GRGDS peptide showed more supramolecular interaction with the PCL-based poly(urea), containing the bisureidobutylene recognition unit, as compared to HMW PCL, lacking this recognition unit.

Chemical and biological properties of supramolecular polymer systems based on oligocaprolactones.

We show that materials with a diverse range of mechanical and biological properties can be obtained using a modular approach by simply mixing different ratios of oligocaprolactones that are either end-functionalized or chain-extended with quadruple hydrogen bonding ureido-pyrimidinone (UPy) moieties. The use of two UPy-synthons allows for easy synthesis of UPy-modified polymers resulting in high yields. Comparison of end-functionalized UPy-polymers with chain-extended UPy-polymers shows that these polymers behave distinctively different regarding their material and biological properties. The end-modified UPy-polymer is rather stiff and brittle due to its high crystallinity. Disks made of this material fractures after subcutaneous implantation. The material shows a low inflammatory response which is accompanied by the formation of a fibrous capsule, reflecting the inertness of the material. The chain-extended UPy-material on the contrary is practically free of crystalline domains and shows clear flexible properties. This material deforms after in-vivo implantation, accompanied with cellular infiltration. By mixing both polymers, materials with intermediate properties concerning their mechanical and biological behaviour can be obtained. Surprisingly, a 20:80 mixture of both polymers with the chain-extended UPy-polymer in excess shows flexible properties without visible deformation upon implantation for 42 days. This mixture, a blend formed by intimate mixing through UPy-UPy interaction, also shows a mild tissue response accompanied with the formation of a thin capsule. The material does not become more crystalline upon implantation. Hence, this mixture might be an ideal scaffold material for soft tissue engineering due to its flexibility and diminished fibrous tissue formation, and illustrates the strength of the modular approach.

Circulating CD34+ progenitor cells modulate host angiogenesis and inflammation in vivo.

Within the phenotypically and functionally heterogeneous group of circulating progenitor cells (CPC), a subclass of cells with vascular repair potential have been identified. These CPC are detected and isolated based on single or combined expression of CD34, CD133 and VEGFR-2, and referred to as endothelial progenitor cells. Here we asked whether CPC subsets defined by single expression of these markers exhibit functional heterogeneity. As functional parameters, we chose the capacity of CPC to differentiate into endothelial cells. Moreover, we studied their role in remodeling by recruitment of inflammatory cells, an aspect that has been little explored. We established an in vivo model in which the intrinsic functional capacity of these human CPC subsets was studied. Human CD34+ CPC, but not CD133+ or VEGFR-2+ CPC, seeded in Matrigel pellets and transplanted subcutaneously in a nude mouse host, contributed little to donor-derived neovascularization. However, host angiogenesis in the Matrigel implant, as demonstrated by the presence of capillaries containing erythrocytes and expressing mouse CD31, was strong in response to implantation of human CD34+ CPC and significantly lower in response to the other two CPC subsets. Moreover, the CD34+ CPC subset was significantly superior to CD133+ CPC and VEGFR-2+ CPC in the recruitment of host monocytes/macrophages. These three CPC populations were further dissected into seven discrete subsets, based on three-parameter flow cytometry analysis of combined expression patterns of CD34, CD133 and VEGFR-2. In conclusion, in our system, CD34+ CPC contribute marginally to neovascularization by differentiation but are potent regulators of the host angiogenic and pro-inflammatory response, suggesting a possible role for these cells in the remodeling of vascular lesions.

Biocompatibility and degradation of aliphatic segmented poly(ester amide)s: in vitro and in vivo evaluation.

Aliphatic segmented poly(ester amide)s, comprising a crystallizable amide phase and a flexible amorphous ester phase, were investigated for potential use in biomedical applications. By varying the amide content and the type of crystallizable amide segments, the polymer's thermal and mechanical properties can readily be tuned. Polymers with 25 and 50 mol % of amide content are noncytotoxic and sustain growth of fibroblasts onto polymer films. The in vitro degradation of these polymers was followed in PBS (pH 7.4) at 37 degrees C up to 7 months. The poly(ester amide)s showed the characteristics of bulk degradation with a gradual decrease in molecular weight and almost no mass loss. The in vivo degradation of these polymers, followed by subcutaneous implantation in rats up to 6 weeks, was slow and similar to the in vitro degradation. The tissue response upon implantation was followed over 6 weeks. A mild foreign-body reaction, characterized by the presence of macrophages, and sporadically a lymphocyte, were observed in the first week of implantation. After 6 weeks the implant site is characterized by fibrous encapsulation with no signs of inflammation. The poly(ester amide)s tested are biocompatible, but their in vitro as well as in vivo degradation is very slow.

A modular and supramolecular approach to bioactive scaffolds for tissue engineering.

Bioactive polymeric scaffolds are a prerequisite for the ultimate formation of functional tissues. Here, we show that supramolecular polymers based on quadruple hydrogen bonding ureido-pyrimidinone (UPy) moieties are eminently suitable for producing such bioactive materials owing to their low-temperature processability, favourable degradation and biocompatible behaviour. Particularly, the reversible nature of the hydrogen bonds allows for a modular approach to gaining control over cellular behaviour and activity both in vitro and in vivo. Bioactive materials are obtained by simply mixing UPy-functionalized polymers with UPy-modified biomolecules. Low-molecular-weight bis-UPy-oligocaprolactones with cell adhesion promoting UPy-Gly-Arg-Gly-Asp-Ser (UPy-GRGDS) and the synergistic UPy-Pro-His-Ser-Arg-Asn (UPy-PHSRN) peptide sequences are synthesized and studied. The in vitro results indicate strong and specific cell binding of fibroblasts to the UPy-functionalized bioactive materials containing both UPy-peptides. An even more striking effect is seen in vivo where the formation of single giant cells at the interface between bioactive material and tissue is triggered.