Secreted protein, acidic, cysteine-rich (SPARC)-related modular calcium binding 1 (SMOC1) has been implicated in the regulation of osteogenic differentiation of human bone marrow mesenchymal stem cells (BMSCs). In this study, we found that a peptide (16 amino acids in length), which is located in the extracellular calcium (EC) binding domain of SMOC1, stimulated osteogenic differentiation of human BMSCs in vitro and calvarial bone regeneration in vivo. Treatment of BMSCs with SMOC1-EC peptide significantly stimulated their mineralization in a dose-dependent manner without changing their rate of proliferation. The expression of osteogenic differentiation marker genes, including type 1 collagen and osteocalcin, also increased in a dose-dependent manner. To examine the effect of the SMOC1-EC peptide on bone formation in vivo, the peptide was covalently immobilized onto hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) particles. X-ray photoelectron spectroscopy analysis showed that the peptide was successfully immobilized onto the surface of HA/β-TCP. Implantation of the SMOC1-EC peptide-immobilized HA/β-TCP particles into mouse calvarial defects and subsequent analyses using microcomputed tomography and histology showed significant bone regeneration compared with that of calvarial defects implanted with unmodified HA/β-TCP particles. Collectively, our data suggest that a peptide derived from the EC domain of SMOC1 induces osteogenic differentiation of human BMSCs in vitro and efficiently enhances bone regeneration in vivo.
Amino Acids ; Animals ; Bone Marrow ; Bone Regeneration ; Calcium ; Ceramics* ; Collagen Type I ; Humans ; In Vitro Techniques ; Mesenchymal Stromal Cells ; Mice ; Miners ; Osteocalcin ; Osteogenesis ; Photoelectron Spectroscopy ; Regeneration* ; X-Ray Microtomography

Full skin auto-grafts are required for reconstruction of skin burns and trauma scars. However, currently available clinical approaches such as sheet skin graft, mesh skin grafts, artificial skin graft, and in vivo skin expansion have limitations due to their potential danger for secondary damage and scar formation at the donor site, and discomfort during skin expansion. We developed an advanced bioreactor system and evaluated its function in skin expansion using porcine full skin. The reactor was designed as a pneumatic cylinder type, was programmed to adjust the pressure and the operating time. The system was composed of culture chamber unit, environmental control unit, and monitoring unit. Skins were expanded at 200 kPa pneumatic force and the expanded skins were analyzed by immunohistochemistry and histology. Furthermore we carried out auto-grafting experiment of the expanded skins in vivo using Yucatan pigs and skins were harvested and histologically analyzed after 8 weeks. The results showed that the bioreactor expanded skins to 160% in 4 hours. Histological analysis of the expanded skins revealed that epidermal cells and dermal fibroblasts were viable and remained integrity. The results of auto-grafting experiment indicated that fibrosis and scars were not detected in the grafted skins. This study demonstrates that the newly developed skin bioreactor enabled to obtain large sized full skin rapidly and successful grating.
Bioreactors* ; Burns ; Cicatrix ; Fibroblasts ; Fibrosis ; Humans ; Immunohistochemistry ; Skin* ; Skin, Artificial ; Swine ; Tissue Donors ; Transplants

In this study, we examined the effect of a combination of fibroblast growth factor-2 (FGF-2) and retinoic acid (RA) on osteoblast and adipocyte lineage commitment and differentiation of human bone marrow mesenchymal stem cells (BMSCs). Pretreatment of human BMSCs with FGF-2 or RA for 5 days followed by osteoblast differentiation induction showed high calcium deposition compared to control. A combination of FGF-2 and RA further induced calcium deposition compared to FGF-2 or RA alone. The enhanced mineral deposition was accompanied with the increased expression of osteoblast differentiation markers, alkaline phosphatase and osteocalcin. On the other hand, FGF-2 pretreatment followed by adipocyte differentiation induction also showed increased formation of lipid droplets in human BMSCs, whereas RA pretreatment suppressed formation of lipid droplets. However, a combination of FGF-2 and RA increased formation of lipid droplets and expression of adipocyte marker genes, including adiponectin, ADIPOQ, FABP4, peroxisome proliferator-activated receptor γ (PPARγ), and C/EBPα. During pretreatment of BMSCs with FGF-2, RA or in combination, the cells expressed similar levels of MSC surface markers such as CD29, CD44, CD90, and CD105, indicating that they maintain stem cell potential. To determine how RA cooperates with FGF-2 in osteoblast and adipocyte lineage commitment, the expression of RA receptors and intracellular lipid-binding proteins was examined. A combination of FGF-2 and RA strongly induced the expression of RA receptor α, β, γ, PPAR β/δ, CRABP-II, and FABP5. Collectively, these results demonstrate that combined pretreatment of human BMSCs with FGF-2 and RA enhances the commitment into osteoblast and adipocyte lineages through modulation of the expression of RA-related genes.
Adipocytes ; Adiponectin ; Alkaline Phosphatase ; Antigens, Differentiation ; Bone Marrow* ; Calcium ; Fibroblast Growth Factor 2* ; Fibroblasts* ; Hand ; Humans ; Lipid Droplets ; Mesenchymal Stromal Cells* ; Miners ; Osteoblasts ; Osteocalcin ; Peroxisome Proliferator-Activated Receptors ; Peroxisomes ; Stem Cells ; Tretinoin*

Mesenchymal stromal cells (MSCs) established by in-vitro adherence culture have been widely utilized for various cell therapeutic trials, but potential heterogeneity that can be caused by preparation methods are poorly characterized. In this study, we show that at least two distinct subsets of MSCs with different adherence to plastic surface exist in human adipose tissue-derived stromal vascular fraction (SVF); while 69% of total colony forming units in SVF adhere to the surface before 3 hrs of plating, 13–17% of colonogenic cells adhered to the surface at later period of 15 hr to 1 week after plating. Of note, the late adherent MSCs exhibited higher self-renewal of colony forming cells and higher proliferating potential with comparable level of osteogenic or adipogenic differentiation potential to the early adherence subsets. Moreover, late adherent cells exhibited distinct pattern of paracrine secretome including higher level secretion of cytokines than the early adherent subsets. Taken together, these results suggest the possibility that distinct adherence properties of MSCs can be another parameter of clonal heterogeneity in the subpopulations of adipose tissue MSCs and that it can be an important factor for optimization of MSC preparation for cell therapeutic trials.
Adipose Tissue* ; Cytokines ; Humans ; Mesenchymal Stromal Cells* ; Plastics* ; Population Characteristics ; Stem Cells

This study aimed to evaluate the in vitro biological effectiveness of chitosan microparticles crosslinked with sodium tripolyphosphate (TPP) in combination with activated pure platelet-rich plasma (aP-PRP) as an injectable composite scaffold for growth factors release, cell proliferation and osteogenic differentiation. Two main novelties were addressed in the field of scaffolds in regenerative medicine: the first is the approach including simultaneously the three vertices of the proliferation triangle formed by the capabilities genic progenitor cells, conductive scaffolds and inductive growth factors, which are provided by platelet rich plasma (PRP); secondly, the approach of an injectable composite scaffolds formed by the fibrin network from aP-PRP and the chitosan microparticles crosslinked with TPP. The microparticles were prepared by vortexing the chitosan and TPP solutions. The ionic crosslinking of chitosan with TPP was made at mass ratios of 2:1, 5:1, and 10:1 at pH 4.0. P-PRP was obtained via the controlled centrifugation of whole blood. The composite scaffolds were prepared by adding the microparticles to immediately activated P-PRP. The results showed that the microparticles had adequate physicochemical and mechanical properties for injection. Furthermore, the microparticles controlled the release of growth factors from P-PRP. The proliferation of human adipose-derived mesenchymal stem cells was lower than in aP-PRP alone but significant at a 2:1 chitosan-TPP mass ratio. Osteogenic differentiation was stimulated at all studied mass ratios, as indicated by the alkaline phosphatase activity. These results offer perspectives for optimizing the composite scaffold, and to prove its potential as an injectable scaffold in regenerative medicine.
Alkaline Phosphatase ; Cell Proliferation ; Centrifugation ; Chitosan ; Fibrin ; Humans ; Hydrogen-Ion Concentration ; In Vitro Techniques* ; Intercellular Signaling Peptides and Proteins ; Mesenchymal Stromal Cells ; Platelet-Rich Plasma* ; Regenerative Medicine ; Sodium ; Stem Cells

Growth factors play multiple and critical roles in wound repair processes. Platelet-derived growth factor (PDGF) is a potent growth factor that is particularly important in the early inflammatory phase of wound healing. In order to extend the half-life of PDGF, polymeric encapsulation is used. In the current study, Poly (lactic-co-glycolic acid) (PLGA) microspheres containing recombinant human (rh) PDGF-BB were prepared to prolong the effectiveness of this growth factor. PLGA microspheres were optimized using a modified w/o/w-double-emulsion/solvent evaporation method by changing the processing conditions of stirring speed and emulsifier (polyvinyl alcohol) concentration. Microspheres prepared using the optimized method released rhPDGF-BB for up to three weeks. An in vitro migration assay showed a significant decrease in the wound area in cells treated with rhPDGF-BB microspheres compared to control cells. These findings demonstrate the potential of rhPDGF-BB encapsulated in microspheres to enhance wound healing.
Half-Life ; Humans* ; In Vitro Techniques ; Intercellular Signaling Peptides and Proteins ; Methods ; Microspheres* ; Platelet-Derived Growth Factor ; Polymers ; Wound Healing* ; Wounds and Injuries*

Cells receive important regulatory signals from their extracellular matrix (ECM) and the physical property of the ECM regulates important cellular behaviors like cell proliferation, migration and differentiation. A large part of tissue formation and regeneration depends on cellular interaction with its ECM. A comprehensive understanding of the mechanistic biochemical pathway of the ECM components is necessary for the design of a biomaterial scaffold for tissue engineering. Depending on the type of tissue, the ECM requirement might be different and this would influence its downstream intracellular cell signaling. Here, we reviewed the ECM and its signaling pathway by discussing: 1) classification of the ECM into hard, elastic and soft tissue based on its physical properties, 2) proliferation and differentiation control of the ECM, 3) roles of membrane receptor and its intracellular regulation factor, 4) ECM remodeling via inside-out signaling. By providing a comprehensive overview of the ECM's role in mechanotransduction and the self-regulatory effect of cells back on the ECM, we hope to provide a better insight of the physical and biochemical cues from the ECM. A sound understanding on the in vivo ECM has implication on the choice of materials and surface coating of biomimetic scaffolds used for tissue regeneration and therapeutics in a cell-free scaffold.
Biocompatible Materials ; Biomimetics ; Cell Proliferation ; Classification ; Cues ; Extracellular Matrix* ; Hope ; Membranes ; Regeneration* ; Tissue Engineering

A major hurdle in engineering thick and laminated tissues such as skin is how to vascularize the tissue. This study introduces a promising strategy for generatingmulti-layering engineered tissue sheets consisting of fibroblasts and endothelial cells co-seeded on highly micro-fibrous, biodegradable polycaprolactone membrane. Analysis of the conditions for induction of the vessels in vivo showed that addition of endothelial cell sheets into the laminated structure increases the number of incorporated cells and promotes primitive endothelial vessel growth. In vivo analysis of 11-layered constructs showed that seeding a high number of endothelial cells resulted in better cell survival and vascularization 4 weeks after implantation.Within one week after implantation in vivo, red blood cells were detected in the middle section of three-layered engineered tissue sheets composed of polycaprolactone/ collagen membranes. Our engineered tissue sheets have several advantages, such as easy handling for cell seeding, manipulation by stacking each layer, a flexible number of cells for next-step applications and versatile tissue regeneration, and automated thick tissue generation with proper vascularization.
Cell Survival ; Collagen ; Endothelial Cells ; Erythrocytes ; Fibroblasts ; Membranes ; Regeneration* ; Skin ; Tissue Engineering

It is controversial whether type I collagen itself can maintain and improve chondrogenic phenotype of chondrocytes in a three-dimensional (3D) environment. In this study, we examined the effect of type I collagen concentration in hydrogel (0.5, 1, and 2 mg/ml) on the growth and phenotype expression of rat chondrocytes in vitro. All collagen hydrogels showed substantial contractions during culture, in a concentration-dependent manner, which was due to the cell proliferation. The cell viability was shown to be the highest in 2 mg/ml collagen gel. The mRNA expression of chondrogenic phenotypes, including SOX9, type II collagen, and aggrecan, was significantly up-regulated, particularly in 1 mg/ml collagen gel. Furthermore, the production of type II collagen and glycosaminoglycan (GAG) content was also enhanced. The results suggest that type I collagen hydrogel is not detrimental to, but may be useful for, the chondrocyte culture for cartilage tissue engineering.
Aggrecans ; Animals ; Cartilage ; Cell Proliferation ; Cell Survival ; Chondrocytes* ; Collagen ; Collagen Type I* ; Collagen Type II ; Hydrogel* ; Hydrogels ; In Vitro Techniques ; Phenotype ; Rats* ; RNA, Messenger ; Tissue Engineering

Expanded polytetrafluoroethylene (ePTFE) polymers do not support endothelialization because of nonconductive characteristics towards cellular attachment. Inner surface modification of the grafts can improve endothelialization and increase the long-term patency rate of the ePTFE vascular grafts. Here we reported a method of inner-surface modification of ePTFE vascular graft with extracellular matrix (ECM) and CD34 monoclonal antibodies (CD34 mAb) to stimulate the adhesion and proliferation of circulating endothelial progenitor cells on ePTFE graft to enhance graft endothelialization. The inner surface of ECM-coated ePTFE grafts were linked with CD34 mAb in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) solution and the physicochemical properties, surface morphology, biocompatibility, and hemocompatibility of the grafts were studied. The hydrophilicity of CD34 mAb-coated graft inner surface was significantly improved. Fourier transform infrared spectroscopy analysis confirmed ECM and CD34 mAb cross-linking in the ePTFE vascular grafts with our method. Scanning electron microscopy analysis showed protein layer covering uniformly on the inner surface of the modified grafts. The cell-counting kit-8 (CCK-8) assay confirmed that the modified graft has no obvious cytotoxicity. The modified graft showed a low hemolytic rate (0.9%) in the direct contact hemolysis test, suggesting the modification improved hemocompatibility of biopolymers. The modification also decreased adhesion of platelets, while significantly increased the adhesion of endothelial cells on the grafts. We conclude that our method enables ePTFE polymers modification with ECM and CD34 mAb, facilitates endothelialization, and inhibits platelet adhesion on the grafts, thus may increase the long-term patency rate of the prosthetic bypass grafts.
Antibodies* ; Antibodies, Monoclonal ; Biopolymers ; Blood Platelets* ; Endothelial Cells ; Endothelial Progenitor Cells ; Extracellular Matrix* ; Hemolysis ; Hydrophobic and Hydrophilic Interactions ; Methods ; Microscopy, Electron, Scanning ; Polymers ; Polytetrafluoroethylene* ; Spectroscopy, Fourier Transform Infrared ; Surface Properties ; Transplants