Vascularization and osteogenesis in ectopically implanted bone tissue-engineered constructs with endothelial and osteogenic differentiated adipose-derived stem cells

BACKGROUND A major problem in the healing of bone defects is insufficient or absent blood supply within the defect.To overcome this challenging problem,a plethora of approaches within bone tissue engineering have been developed recently.Bearing in mind that the interplay of various diffusible factors released by endothelial cells(ECs)and osteoblasts(OBs)have a pivotal role in bone growth and regeneration and that adjacent ECs and OBs also communicate directly through gap junctions,we set the focus on the simultaneous application of these cell types together with platelet-rich plasma(PRP)as a growth factor reservoir within ectopic bone tissue engineering constructs.AIM To vascularize and examine osteogenesis in bone tissue engineering constructs enriched with PRP and adipose-derived stem cells(ASCs)induced into ECs and OBs.METHODS ASCs isolated from adipose tissue,induced in vitro into ECs,OBs or just expanded were used for implant construction as followed:BPEO,endothelial and osteogenic differentiated ASCs with PRP and bone mineral matrix;BPUI,uninduced ASCs with PRP and bone mineral matrix;BC(control),only bone mineral matrix.At 1,2,4 and 8 wk after subcutaneous implantation in mice,implants were extracted and endothelial-related and bone-related gene expression were analyzed,while histological analyses were performed after 2 and 8 wk.RESULTS The percentage of vascularization was significantly higher in BC compared to BPUI and BPEO constructs 2 and 8 wk after implantation.BC had the lowest endothelial-related gene expression,weaker osteocalcin immunoexpression and Spp1 expression compared to BPUI and BPEO.Endothelial-related gene expression and osteocalcin immunoexpression were higher in BPUI compared to BC and BPEO.BPEO had a higher percentage of vascularization compared to BPUI and the highest CD31 immunoexpression among examined constructs.Except Vwf,endothelial-related gene expression in BPEO had a later onset and was upregulated and well-balanced during in vivo incubation that induced late onset of Spp1 expression and pronounced osteocalcin immunoexpression at 2 and 8 wk.Tissue regression was noticed in BPEO constructs after 8 wk.CONCLUSION Ectopically implanted BPEO constructs had a favorable impact on vascularization and osteogenesis,but tissue regression imposed the need for discovering a more optimal EC/OB ratio prior to considerations for clinical applications.     

In vitro and in vivo differentiation of human umbilical cord derived stem cells into endothelial cells

The successful use of tissue-engineered transplants is hampered by the need for vascularization. Recent advances have made possible the using of stem cells as cell sources for therapeutic angiogenesis, including the vascularization of engineered tissue grafts. The goal of this study was to examine the endothelial potential of human umbilical cord-derived stem (UCDS) cells. UCDS cells were initially characterized and differentiated in an endothelial differentiation medium containing VEGF and bFGF. Differentiation into endothelial cells was determined by acetylated low-density lipoprotein incorporation and expression of endothelial-specific proteins, such as PECAM and CD34. In vivo, the transplanted UCDS cells were sprouting from local injection and differentiated into endothelial cells in a hindlimb ischemia mouse model. These findings indicate the presence of a cell population within the human umbilical cord that exhibits characteristics of endothelial progenitor cells. Therefore, human umbilical cord might represent a source of stem cells useful for therapeutic angiogenesis and re-endothelialization of engineered tissue grafts.     

Transplantation of adipose derived stem cells for peripheral nerve regeneration in sciatic nerve defects of the rat

Tissue engineering approaches for promoting the repair of peripheral nerve injuries have focused on cell-based therapies involving Adipose-derived stem cells (ASCs). The authors evaluated the effects of undifferentiated ASCs and of neurally differentiated ASCs on the regenerating abilities of peripheral nerves. We hope that this would demonstrate the feasibility of using adipose derived stem cells for peripheral nerve regeneration and provide clues regarding the use of adipose- derived stem cells. ASCs were isolated and cultured. Then the cells were cultured with neuronal induction agents for neural differentiation. ASCs and neurally differentiated ASCs were transplanted into sciatic nerve defects. After 12 weeks, the number and diameter of the myelinated fibers were measured and nerve conduction study was done. The extent of regeneration of myelinated fibers in the neurally differentiated ASCs transplanted group was greater than that in the ASCs transplanted group or the control group. However, thickness of myelin sheath and diameter of nerve fibers in the ASCs transplanted group were greater than those in the neutrally differentiated ASCs transplanted group or the control group. Nerve conduction study showed good recovery in the neurally differentiated ASCs transplanted groups. Muscles can atrophy and contract if denervation has started. It would be difficult to recover muscle function even if the nerve was reinnervated. Therefore, although neurally differentiated ASCs were found to have a greater functional effect than non-differentiated ASCs, time constraint is important when considering a method of ASCs transplantation.     

小鼠骨髓内皮细胞条件培养液复合FL及TPO对HPP—CFC及CFU—GM增殖的影响

通过传代培养小鼠骨髓内皮细胞,收集无血清条件培养液（ECM）,并经超滤得到大于10kD的浓缩液,分别观察ECM和大于10kD的浓缩液复合flt3ligand(FL)及thrombopoietin(TPO)对体外培养HPP-CFC、CFU-GM的影响,结果表明：ECM或大于10kD的浓缩液对HPP-CFC、CFU-GM的生长均有支持作用;FL或/和TPO与ECM或大于10kD的浓缩液合用能加强对HPP-CFC、CFU-GM生长的刺激作用;FL加TPO与ECM或大于10kD的浓缩液合用对HPP-CFC、CFU-GM生长的刺激作用更加明显;选择FL和TPO特异性的引物,用RT-PCR技术未能检测到小鼠骨髓内皮细胞有FL和TPOmRNA的表达。     

Effect of different cell sheet ECM microenvironment on the formation of vascular network

The repair and reconstruction of large bone defects remains as a significant clinical challenge mainly due to the insufficient vascularization. The prefabrication of vascular network based on cell sheet technique brings a promising potential for sufficient vascularization due to rich extracellular matrix (ECM) of cell sheets. However, the effect of different cell sheet ECM micro-environment on the formation of a vascular network has not been well understood. Here our goal is to study the effect of different cell sheets on the formation of a vascular network. First we cultured human bone marrow mesenchymal stem cells (hBMSCs) under two culture conditions to obtain osteogenic differentiated cell sheet (ODCS) and undifferentiated cell sheet (UDCS), respectively. Then the human umbilical vein endothelial cells (HUVECs) were seeded onto the surface of the two sheets at different seeding densities to fabricate pre-vascularized cell sheets. Our results indicated that the two sheets facilitated the alignment of HUVECs and promoted the formation of vascular networks. Quantitative analysis showed that the number of networks in ODCS was higher than that in the UDCS. The ECM of the two sheets was remodeled and rearranged during the tubulogenesis process. Furthermore, results showed that the optimal seeding density of HUVECs was 5 × 10⁴ cell/cm². In summary, these results suggest that the vascularized ODCS has a promising potential to construct pre-vascularized tissue for bone repair.     

Altered Metabolic and Stemness Capacity of Adipose Tissue-Derived Stem Cells from Obese Mouse and Human

Adipose stem cells (ASCs) are an appealing source of cells for therapeutic intervention; however, the environment from which ASCs are isolated may impact their usefulness. Using a range of functional assays, we have evaluated whether ASCs isolated from an obese environment are comparable to cells from non-obese adipose tissue. Results showed that ASCs isolated from obese tissue have a reduced proliferative ability and a loss of viability together with changes in telomerase activity and DNA telomere length, suggesting a decreased self-renewal capacity. Metabolic analysis demonstrated that mitochondrial content and function was impaired in obese-derived ASCs resulting in changes in favored oxidative substrates. These findings highlight the impact of obesity on adult stem properties. Hence, caution should be exercised when considering the source of ASCs for cellular therapies since their therapeutic potential may be impaired.     

Sphingosine-1-phosphate promotes the differentiation of adipose-derived stem cells into endothelial nitric oxide synthase (eNOS) expressing endothelial-like cells

Background

Adipose tissue provides a readily available source of autologous stem cells. Adipose-derived stem cells (ASCs) have been proposed as a source for endothelial cell substitutes for lining the luminal surface of tissue engineered bypass grafts. Endothelial nitric oxide synthase (eNOS) is a key protein in endothelial cell function. Currently, endothelial differentiation from ASCs is limited by poor eNOS expression. The goal of this study was to investigate the role of three molecules, sphingosine-1-phosphate (S1P), bradykinin, and prostaglandin-E1 (PGE1) in ASC endothelial differentiation. Endothelial differentiation markers (CD31, vWF and eNOS) were used to evaluate the level of ASCs differentiation capability.

Results

ASCs demonstrated differentiation capability toward to adipose, osteocyte and endothelial like cell phenotypes. Bradykinin, S1P and PGE were used to promote differentiation of ASCs to an endothelial phenotype. Real-time PCR showed that all three molecules induced significantly greater expression of endothelial differentiation markers CD31, vWF and eNOS than untreated cells. Among the three molecules, S1P showed the highest up-regulation on endothelial differentiation markers. Immunostaining confirmed presence of more eNOS in cells treated with S1P than the other groups. Cell growth measurements by MTT assay, cell counting and EdU DNA incorporation suggest that S1P promotes cell growth during ASCs endothelial differentiation. The S1P1 receptor was expressed in ASC-differentiated endothelial cells and S1P induced up-regulation of PI3K.

Conclusions

S1P up-regulates endothelial cell markers including eNOS in ASCs differentiated to endothelial like cells. This up-regulation appears to be mediated by the up-regulation of PI3K via S1P1 receptor. ASCs treated with S1P offer promising use as endothelial cell substitutes for tissue engineered vascular grafts and vascular networks.     

The cardioprotective effect of mesenchymal stem cells is mediated by IGF-I and VEGF

Emerging evidence suggests that adipose tissue-derived stem cells (ASCs) can be used for the treatment of ischemic heart diseases. However, the mechanisms underlying their therapeutic effects have not been clearly defined. In this study cytokines released by ASCs were detected by ELISA and pro-angiogenic effects were assessed by tube formation assay. To define the anti-apoptotic effect of ASCs, neonatal rat cardiomyocytes were subjected to hypoxia condition in a co-culture system. Our data show that ASCs secrete significant amounts of VEGF (810.65 ± 56.92 pg/μg DNA) and IGF-I (328.33 ± 22.7 pg/μg DNA). Cardiomyocytes apoptosis was significantly prevented by ASCs and 62.5% of the anti-apoptotic effect was mediated by IGF-I and 34.2% by VEGF. ASCs promoted endothelial cell tube formation by secreting VEGF. In conclusion we demonstrated that ASCs have a marked impact on anti-apoptosis and angiogenesis and helps to explain data of stem cells benefit without transdifferentiation.     

Improving the integrity of three-dimensional vascular patterns by poly(ethylene glycol) conjugation

Development of functional tissue-engineering constructs may require that multiple cell types be organized in controlled three-dimensional (3-D) microarchitectures with proper nutrient diffusion and vascularization. In the past few years, a variety of microscale techniques have demonstrated the ability to control protein and cell attachment in defined patterns. Nevertheless, maintenance of these patterns over time has been a significant challenge due to nonspecific protein adsorption and cell migration. To this end, we have investigated the effectiveness of poly(ethylene glycol) (PEG) thin films in maintaining the integrity of 3-D cellular patterns, using human umbilical vein endothelial cells (HUVEC) as a model system. These HUVEC constructs were created using extracellular matrix (ECM)-based microfluidic patterning. Our results indicated that PEG-conjugated substrates improve cell pattern integrity as compared to control silicon. The compliance multifactor (a measure of pattern integrity; higher value means lower pattern integrity) was about 3.66 +/- 0.29 on day 5 for PEG-conjugated surfaces, compared with 8.23 +/- 0.42 for control surfaces ECM-based microfluidic patterning coupled with stable PEG-conjugated surfaces may serve as a vital tool for vascularized tissue engineering.     

Engineering a bilayered hydrogel to control ASC differentiation

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in vivo. An area of research that holds promise in regenerative medicine is the combinatorial use of novel biomaterials and stem cells. A fundamental strategy in the field of tissue engineering is the use of three-dimensional scaffold (e.g., decellularized extracellular matrix, hydrogels, micro/nano particles) for directing cell function. This technology has evolved from the discovery that cells need a substrate upon which they can adhere, proliferate, and express their differentiated cellular phenotype and function. More recently, it has also been determined that cells not only use these substrates for adherence, but also interact and take cues from the matrix substrate (e.g., extracellular matrix, ECM). Therefore, the cells and scaffolds have a reciprocal connection that serves to control tissue development, organization, and ultimate function. Adipose-derived stem cells (ASCs) are mesenchymal, non-hematopoetic stem cells present in adipose tissue that can exhibit multi-lineage differentiation and serve as a readily available source of cells (i.e. pre-vascular endothelia and pericytes). Our hypothesis is that adipose-derived stem cells can be directed toward differing phenotypes simultaneously by simply co-culturing them in bilayered matrices. Our laboratory is focused on dermal wound healing. To this end, we created a single composite matrix from the natural biomaterials, fibrin, collagen, and chitosan that can mimic the characteristics and functions of a dermal-specific wound healing ECM environment.     

Galantamine and carbon monoxide protect brain microvascular endothelial cells by heme oxygenase-1 induction

Galantamine, a reversible inhibitor of acetylcholine esterase (AChE), is a novel drug treatment for mild to moderate Alzheimer’s disease and vascular dementia. Interestingly, it has been suggested that galantamine treatment is associated with more clinical benefit in patients with mild-to-moderate Alzheimer disease compared to other AChE inhibitors. We hypothesized that the protective effects of galantamine would involve induction of the protective gene, heme oxygenase-1 (HO-1), in addition to enhancement of the cholinergic system. Brain microvascular endothelial cells (mvECs) were isolated from spontaneous hypertensive rats. Galantamine significantly reduced H₂O₂-induced cell death of mvECs in association with HO-1 induction. These protective effects were completely reversed by nuclear factor-κB (NF-κB) inhibition or HO inhibition. Furthermore, galantamine failed to induce HO-1 in mvECs which lack inducible nitric oxide synthase (iNOS), supplementation of a nitric oxide (NO) donor or iNOS gene transfection on iNOS-deficient mvECs resulted in HO-1 induction with galantamine. These data suggest that the protective effects of galantamine require NF-κB activation and iNOS expression, in addition to HO-1. Likewise, carbon monoxide (CO), one of the byproducts of HO, up-regulated HO-1 and protected mvECs from oxidative stress in a similar manner. Our data demonstrate that galantamine mediates cytoprotective effects on mvECs through induction HO-1. This pharmacological action of galantamine may, at least in part, account for the superior clinical efficacy of galantamine in vascular dementia and Alzheimer disease.     

Osteoblasts extracellular matrix induces vessel like structures through glycosylated collagen I

Extracellular matrix (ECM) plays a fundamental role in angiogenesis affecting endothelial cells proliferation, migration and differentiation. Vessels-like network formation in vitro is a reliable test to study the inductive effects of ECM on angiogenesis. Here we utilized matrix deposed by osteoblasts as substrate where the molecular and structural complexity of the endogenous ECM is preserved, to test if it induces vessel-like network formation by endothelial cells in vitro. ECM is more similar to the physiological substrate in vivo than other substrates previously utilized for these studies in vitro. Osteogenic ECM, prepared in vitro from mature osteoblasts at the phase of maximal deposition and glycosylation of collagen I, induces EAhy926, HUVEC, and HDMEC endothelial cells to form vessels-like structures and promotes the activation of metalloproteinase-2 (MMP-2); the functionality of the p-38/MAPK signaling pathway is required. Osteogenic ECM also induces a transient increase of CXCL12 and a decrease of the receptor CXCR4. The induction of vessel-like networks is dependent from proper glycosylation of collagens and does not occur on osteogenic ECMs if deglycosylated by -galactosidase or on less glycosylated ECMs derived from preosteoblasts and normal fibroblasts, while is sustained on ECM from osteogenesis imperfecta fibroblasts only when their mutation is associated with over-glycosylation of collagen type I. These data support that post-translational glycosylation has a role in the induction in endothelial cells in vitro of molecules conductive to self-organization in vessels-like structures.     

Comparison of adult stem cells derived from multiple stem cell niches

Objectives

Adult stem cells (ASCs) have great potential for tissue regeneration; however, comparative studies of ASCs from different niches are required to understand the characteristics of each population for their potential therapeutic uses.

Results

We compared the proliferation, stem cell marker expression, and differentiation potential of ASCs from bone marrow, skin dermis, and adipose tissue. ASCs from bone marrow and skin dermis showed 50–100 % increased proliferation in comparison to the ASCs from adipose tissues. Furthermore, ASCs from each stem cell niche showed differential expression of stem cell marker genes, and preferentially differentiated into cell types of their tissue of origin.

Conclusion

Different characters of each ASC might be major factors for their effective use for therapeutics and tissue regeneration.

     

Cell interaction with the extracellular matrices produced by endothelial cells and fibroblasts

The extracellular matrices (ECM) produced by cultured bovine corneal endothelial cells and chick embryo fibroblasts were compared for their induction of cell attachment, proliferation and differentiation. The corneal endothelial ECM (cECM) induced a comparable and rapid attachment and flattening of both human Ewing's sarcoma and colon carcinoma cells which utilize fibronectin and laminin as adhesive glycoproteins, respectively. In contrast, the ECM produced by fibroblasts (fECM) readily supported the attachment and flattening of Ewing's sarcoma cells but had only a small effect on the carcinoma cells. Vascular endothelial cells were stimulated to proliferate by both types of matrices, but to a lesser extent by the fECM. In contrast, the formation of a closely apposed, non-overlapping and contact-inhibited endothelial cell monolayer was only dictated by the cECM. Vascular endothelial cells cultured on fECM grew on top of each other and incorporated [3H]thymidine even late at confluency. Neurite outgrowth (ciliary ganglion cells) and network formation (adult rat oligodendrocytes) were promoted by both types of matrices but in a more consistent manner with the cECM. It is likely that the small amounts of laminin deposited by chick embryo fibroblasts into their ECM are responsible for its efficient induction of neurite outgrowth and for the limited degree of carcinoma cell attachment and flattening. It is thus demonstrated that differences in chemical composition and supramolecular arrangement between cECM and fECM result not only in differences in the attachment, spreading and proliferative responses of cells but also in the expression of their characteristic morphological appearance and differentiated functions.     

Advancements in adipose-derived stem cell therapy for skin fibrosis

Pathological scarring and scleroderma, which are the most common conditions of skin fibrosis, pathologically manifest as fibroblast proliferation and extracellular matrix (ECM) hyperplasia. Fibroblast proliferation and ECM hyperplasia lead to fibrotic tissue remodeling, causing an exaggerated and prolonged wound-healing response. The pathogenesis of these diseases has not been fully clarified and is unfortunately accompanied by exceptionally high medical needs and poor treatment effects. Currently, a promising and relatively low-cost treatment has emerged-adipose-derived stem cell (ASC) therapy as a branch of stem cell therapy, including ASCs and their derivatives-purified ASC, stromal vascular fraction, ASC-conditioned medium, ASC exosomes, etc., which are rich in sources and easy to obtain. ASCs have been widely used in therapeutic settings for patients, primarily for the defection of soft tissues, such as breast enhancement and facial contouring. In the field of skin regeneration, ASC therapy has become a hot research topic because it is beneficial for reversing skin fibrosis. The ability of ASCs to control profibrotic factors as well as anti-inflammatory and immunomodulatory actions will be discussed in this review, as well as their new applications in the treatment of skin fibrosis. Although the long-term effect of ASC therapy is still unclear, ASCs have emerged as one of the most promising systemic antifibrotic therapies under development.     

Bone regeneration in a rabbit ulna defect model: use of allogeneic adipose-derivedstem cells with low immunogenicity

Tissue engineering provides new potential treatments for the repair of bone defects. Bone-marrow-derived mesenchymal stem cells (BMSCs) represent an attractive cell source for therapeutic applications involving tissue engineering, although disadvantages, such as pain of harvest and low proliferation efficiency, are major limitations to the application of BMSCs in the clinic. Adipose-derived stem cells (ASCs) with their multilineage potential and satisfactory proliferation potential can be induced into the osteogenic lineage in vitro and can be anchored onto suitable scaffolds as seed cells to repair bone defects successfully in an autologous setting. Previous studies have indicated that both undifferentiated BMSCs and ASCs exhibit immunosuppression and immunoprivilege properties. We compare the immuno-function of undifferentiated and osteo-differentiated ASCs in vitro and explore the feasibility of applying allogeneic ASCs to the repair of ulnar bone defects in the rabbit model. Our study demonstrates that allogeneic osteogenic differentiated ASCs maintain low immunogenicity and negative immunomodulation. The allogeneic osteogenic differentiated ASCs combined with demineralized bone matrix successfully regenerate ulnar bone defects in rabbits without immunosuppressive therapies.     

Effects of transforming growth factor-beta1 on cell motility, collagen gel contraction, myofibroblastic differentiation, and extracellular matrix expression of human adipose-derived stem cell

Human adipose-derived stem cells (ASCs) are adult pluripotent stem cells, and their usefulness in plastic surgery has garnered attention in recent years. Although, there have been expectations that ASCs might function in wound repair and regeneration, no studies to date have examined the role of ASCs in the mechanism that promotes wound-healing. Transforming growth factor-beta1 (TGF-β1) is a strong candidate cytokine for the triggering of mesenchymal stem cell migration, construction of extracellular matrices, and differentiation of ASCs into myofibroblasts. Cell proliferation, motility, and differentiation, as well as extracellular matrix production, play an important role in wound-healing. We have evaluated the capacity of ASCs to proliferate and their potential to differentiate into phenotypic myofibroblasts, as well as their cell motility and collagen gel contraction ability, when cultured with TGF-β1. Cell motility was analyzed using a wound-healing assay. ASCs that differentiated into myofibroblasts expressed the gene for alpha-smooth muscle actin, and its protein expression was detected immunohistochemically. The extracellular matrix expression in ASCs was evaluated using real-time RT-PCR. Based on the results, we conclude that human ASCs have the potential for cell motility, extracellular matrix gene expression, gel contraction, and differentiation into myofibroblasts and, therefore, may play an important role in the wound-healing process.     

Immunofluorescent localization of type IV collagen and laminin during endochondral bone differentiation and regulation by pituitary growth hormone

Vascularization and the influence of growth hormone on this process were studied during endochondral bone differentiation. Vascular invasion was monitored by immunofluorescent localization of two vascular basement membrane proteins, type IV collagen and laminin, a recently described glycoprotein. In addition, endothelial cell invasion was identified by localization of Factor VIII. New bone formation was induced by subcutaneous implantation of a coarse powder of demineralized rat bone matrix. On days 1 through 9, no vascular elements were detected in the plaque. Mesenchymal cells appeared on day 3, proliferated, and differentiated into cartilage on day 7, while the capillaries proliferated at the periphery of the plaque. Beginning on day 9 with capillary incursion into the center of the plaque, type IV collagen, laminin, and Factor VIII were localized in the invading vascular endothelial cells. Type IV collagen and laminin appeared synchronously in the capillary basement membranes and later in the endothelial lining of cavernous sinusoids. Their distribution pattern was identical. The vascular invasion was prominent by day 14. In hypophysectomized rats, cartilage differentiated normally but vascularization was delayed and reduced. Bone formation was scanty as indicated by ⁴⁵Ca incorporation. Administration of bovine growth hormone to hypophysectomized recipients restored vascularization and bone formation to the level observed in controls.