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Chemical modification of gelatin using epigallocatechin gallate (EGCG) promotes bone formation in vivo. However, further improvements are required to increase the mechanical strength and bone-forming ability of fabricated EGCG-modified gelatin sponges (EGCG-GS) for practical applications in regenerative therapy. In the present study, we investigated whether vacuum heating-induced dehydrothermal cross-linking of EGCG-GS enhances bone formation in critical-sized rat calvarial defects. The bone-forming ability of vacuum-heated EGCG-GS (vhEGCG-GS) and other sponges was evaluated by micro-computed tomography and histological staining. The degradation of sponges was assessed using protein assays, and cell morphology and proliferation were verified by scanning electron microscopy and immunostaining using osteoblastic UMR106 cells in vitro. Four weeks after the implantation of sponges, greater bone formation was detected for vhEGCG-GS than for EGCG-GS or vacuum-heated gelatin sponges (dehydrothermal cross-linked sponges without EGCG). In vitro experiments revealed that the relatively low degradability of vhEGCG-GS supports cell attachment, proliferation, and cell-cell communication on the matrix. These findings suggest that vacuum heating enhanced the bone forming ability of EGCG-GS, possibly via the dehydrothermal cross-linking of EGCG-GS, which provides a scaffold for cells, and by maintaining the pharmacological effect of EGCG., Competing Interests: The authors declare no conflict of interest.

We evaluated the effect of porous alpha-tricalcium phosphate (α-TCP) with immobilized basic fibroblast growth factor (bFGF) on periodontal regeneration in a canine model of 2-wall periodontal defects. Identical bone defects were made in the canine mandible; six defects in each animal were filled with porous α-TCP with bFGF bound via heparin (bFGF group), and the remaining defects were filled with unmodified porous α-TCP (control group). Micro-computed tomography and histological evaluation were performed at 2, 4, and 8 weeks post-implantation. The bone mineral content of the bFGF group was higher than that of the control group at 2 and 4 weeks (p < 0.05). Histological evaluation at 2 weeks post-implantation revealed degradation of the porous α-TCP, and bone had formed on the surface of α-TCP particles in the bFGF group. Some of these collagen fibers connected the newly formed cementum with the alveolar bone, revealing the formation of new periodontal ligaments with Sharpey's fibers. At 8 weeks, continuous cortical bone with a Haversian structure covered the top of the bone defects in the bFGF group. These findings indicate that porous α-TCP with immobilized bFGF could promote periodontal regeneration at the early regeneration phase in a canine model of 2-wall periodontal defects.

Introduction: We evaluated the effects of synthesized collagen model polypeptides consisting of a proline-hydroxyproline-glycine (poly(PHG)) sequence combined with porous alpha-tricalcium phosphate (α-TCP) particles on bone formation in a canine tibia defect model., Materials and Methods: The porous α-TCP particles were mixed with a poly(PHG) solution, and the obtained sponge was then cross-linked and characterized by x-ray diffraction and scanning electron microscopy. Tibia defects were analyzed in 12 healthy beagles using microcomputed tomography and histological evaluation., Results: At 2 and 4 weeks, the volume density of new bone was higher in the poly(PHG)/α-TCP group than in poly(PHG) alone group (P < 0.05); however, there was no difference at 8 weeks (P > 0.05). Histological evaluation at 4 weeks after implantation revealed that the poly(PHG) had degraded, and newly formed bone was present on the surface of the α-TCP particles. At 8 weeks, continuous cortical bone formation with a Haversian structure covered the top of the bone defects in both groups., Conclusion: This study demonstrates that the composite created using porous α-TCP particles and poly(PHG) is sufficiently adaptable for treating bone defects.

The objective of this study was to investigate the feasibility of biodegradable gelatin-β-tricalcium phosphate (β-TCP) composites as a cell scaffold and controlled-release carrier of basic fibroblast growth factor (bFGF) suitable for inducing bone regeneration at a segmental bone defect. The composite of gelatin sponge and β-TCP granules had an interconnected pore structure with an average size of 340 µm. The composite provided the controlled release of bFGF over 2 weeks. Segmental, critical-sized, bone defects of 20 mm length were created in the ulnas of New Zealand white rabbits and the gelatin-β-TCP composites, with or without incorporated bFGF, were implanted into the defects. Bone regeneration and β-TCP resorption profiles were evaluated by microcomputed tomography scanner analysis and haematoxylin and eosin staining. The composites incorporating bFGF promoted significantly higher bone regeneration at the defect site as compared to the bFGF-free composites. The controlled release of biologically active bFGF from the composites may possibly be achieved through the biodegradation of the composites, resulting in the promotion of bone regeneration. We conclude that the biodegradable gelatin-β-TCP composite is a promising scaffold for bone regeneration that enables the controlled release of bFGF., (Copyright © 2012 John Wiley & Sons, Ltd.)

We evaluated the effects on bone formation of combining synthesized collagen model polypeptides consisting of a Pro-Hyp-Gly [poly(PHG)] sequence and alpha-tricalcium phosphate (α-TCP) particles with various median sizes (large: 580.8 μm; small: 136.2 μm; or large and small mixed: 499.3 μm) in a skull defect model in mini-pigs. Quantitative image analyses for the volume density (VD) of new bone revealed that the VD in each α-TCP group was significantly higher than that in the poly(PHG) control group, with the mixed group showing the highest VD among all the groups at 4 weeks after implantation. Histological assessments revealed that the small α-TCP particles were almost completely degraded at 8 weeks. At 12 weeks, all sizes of α-TCP particles were completely degraded and remodeling of the lamellar bone was observed. The present findings suggest that particle size may influence the success of bone formation in defects.

To date, there has been no study on the development of novel regimens based on the following tissue engineering principles: seeding and culturing mesenchymal stem cells (MSCs) on a scaffold before surgery or injecting cultured MSCs into a scaffold during surgery. The purpose of this study was to assess the in vivo osteogenic ability of scaffold/MSCs implanted beneath the periosteum of the cranial bone of rats in three different sample groups: one in which MSCs were pre-seeded and cultured on a scaffold to produce the 3-D woven fabric scaffold/MSC composite using osteo-lineage induction medium, one in which cultured MSCs produced by osteo-lineage induction in cell cultivation flasks were injected into a scaffold during surgery and a control group, in which only the 3-D woven fabric scaffold was implanted. The results indicate that pre-seeding MSCs on a scaffold leads to a higher osteogenic ability than injecting cultured MSCs into a scaffold during surgery.

A novel, injectable bone tissue engineering material was developed that consisted of beta-tricalcium phosphate (beta-TCP) beads as the solid phase and alginate as the gel phase. To prepare the instantaneously formed composite scaffold, an aqueous calcium chloride solution was dried on the surface of beta-TCP beads and crosslinked with an alginic acid sodium solution, thereby forming stable beta-TCP beads and alginate gel which were injectable via a syringe. This biodegradable composite was a three-dimensional (3D) material that could be used as an injectable scaffold for bone tissue engineering. In particular, the composite with 2.0 wt% alginate concentration exhibited a compressive strength of 69 kPa in dry conditions, which was significantly higher than that exhibited by 1.0 wt%. Furthermore, mesenchymal stem cells (MSC) were 3D-cultured within the composite and then investigated for osteogenic markers. MSC-loaded composite was subjected to scanning electron microscope (SEM) examination and implanted subcutaneously for in vivo experiment. Results showed that the scaffold provided support for osteogenic differentiation. In light of the encouraging results obtained, this novel injectable composite material may be useful for bone tissue engineering.

Synthetic biomaterials have been developed and used for bone grafting. Here, we developed a biodegradable sponge composite for bone tissue engineering by combining beta-tricalcium phosphate (beta-TCP) and collagen. In addition, we sought to determine the optimal beta-TCP granules/collagen ratio by evaluating and bone formation in vivo. Porous beta-TCP granules were mixed with atelocollagen hydrochloride solution at various ratios--0.02, 0.05, 0.1, and 0.2 g/mL. The resultant mixtures were freeze-dried and subjected to dehydrothermal treatment in vacuo. The final composites obtained were designated beta-TCP/collagen sponge composites (beta-TCP/CS). Through compression testing, it was found that the stress values for beta-TCP/CS (0.2 g/mL) were higher than those of the other three composites over the whole strain range. Histological evaluation at four weeks after implantation revealed that the collagen sponge had degraded and newly formed bone was present on the surface of the beta-TCP granules. At 12 weeks, the beta-TCP granules were completely degraded and remodeling of the lamellar bone was observed.

Bone reconstruction in the maxillofacial region typically relies on autologous bone grafting, which presents challenges, including donor site complications and graft limitations. Recent advances in tissue engineering have identified highly pure and proliferative dedifferentiated fat cells (DFATs) as promising alternatives. Herein, we explored the capacity for osteoblast differentiation and the osteoinductive characteristics of extracellular vesicles derived from DFATs (DFAT-EVs). DFATs were isolated from human buccal fat pads, cultured to confluency, and placed in either a standard or osteogenic induction medium. After culturing for 3 days, the conditioned medium was used to generate EVs using the size-exclusion chromatography and concentration filter method. Characterization of DFAT-EVs revealed typical EV morphology and positive markers (CD9 and CD63), with no differences between the two groups. In vitro assays demonstrated that EVs derived from the osteogenic induction medium (OI-EVs) significantly increased alkaline phosphatase activity and osteogenesis-related genes (Runx2 and collagen type I) compared to control EVs. Next-generation sequencing identified differentially expressed miRNAs, and gene ontology analysis suggested pathways involved in osteoblast differentiation. Isolating DFATs from buccal fat pads under osteogenic induction conditions offers a procedure confined to the oral cavity, eliminating the need for harvesting from other sites. Thus, DFAT-EVs hold promise for promoting bone regeneration in maxillofacial applications. [ABSTRACT FROM AUTHOR]

This study aimed to develop a novel culture method for rat adipose-derived stem cells (rADSC) and evaluate their osteogenic potential. The rADSC cultured in xeno-free culture medium (XF-rADSCs) or conventional culture medium containing fetal bovine serum (FBS-rADSCs) were combined with micropieces of xeno-free recombinant collagen peptide to form 3-dimensional aggregates (XF-rADSC-CellSaic or FBS-rADSC-CellSaic). Both FBS-rADSC and XF-ADSC in CellSaic exhibited multilineage differentiation potential. Compared to FBS-rADSC-CellSaic, XF-rADSC-CellSaic accelerated and promoted osteogenic differentiation in vitro. When transplanted into rat mandibular congenital bone defects, the osteogenically differentiated XF-rADSC-CellSaic induced regeneration of bone tissue with a highly maturated structure compared to FBS-rADSC-CellSaic. In conclusion, XF-rADSC-CellSaic is a feasible 3-dimensional platform for efficient bone formation. [ABSTRACT FROM AUTHOR]

Mechanical and resorbable scaffolds are in high demand for stem cell-based regenerative medicine, to treat refractory bone defects in craniofacial abnormalities and injuries. Multipotent progenitor cells, such as dedifferentiated fat (DFAT) cells, are prospective sources for regenerative therapies. Herein, we aimed to demonstrate that a composite gelatin sponge (α-TCP/GS) of alfa-tricalcium phosphate (α-TCP) mixed with gelatin scaffolds (GS), with/without DFATs, induced bone regeneration in a rat calvarial defect model in vivo. α-TCP/GS was prepared by mixing α-TCP and 2% GS using vacuum-heated methods. α-TCP/GS samples with/without DFATs were transplanted into the model. After 4 weeks of implantation, the samples were subjected to micro-computed tomography (μ-CT) and histological analysis. α-TCP/GS possessed adequate mechanical strength; α-TCP did not convert to hydroxyapatite upon contact with water, as determined by X-ray diffraction. Moreover, stable α-TCP/GS was formed by electrostatic interactions, and verified based on the infrared peak shifts. μ-CT analyses showed that bone formation was higher in the α-TCP/GS+ DFAT group than in the α-TCP/GS group. Therefore, the implantation of α-TCP/GS comprising DFAT cells enhanced bone regeneration and vascularization, demonstrating the potential for healing critical-sized bone defects. [ABSTRACT FROM AUTHOR]

Abstract: Purpose: Both atelocollagen and alpha-tricalcium phosphate (α-TCP) particles are widely applied as bone graft materials. In this study, we combined atelocollagen and porous α-TCP particles to form an α-TCP/atelocollagen composite sponge (α-TCP/CS), and we evaluated its effects on bone regeneration in rat calvarial defects. Materials and methods: α-TCP granules were mixed with a collagen solution. The mixture was poured into plastic molds and then frozen to −80°C and freeze-dried for 24h. The composites were then cross-linked in vacuo at 140°C for 24h. The obtained composites were characterized by XRD and SEM and observed using micro-CT and histological analysis. Results: In vivo micro-CT images at 2, 4, and 6 weeks after surgery showed that in the α-TCP/CS group, the bone volume, bone mineral density, and bone mineral content were higher than those in the groups with only atelocollagen and without scaffolds at 6 weeks after surgery. Histological analysis showed that α-TCP was completely absorbed, and new bone that was continuous along the original bone was observed. Conclusion: This study demonstrated that composite sponges created using porous α-TCP particles and atelocollagen were sufficiently adaptable for treating bone defects. [Copyright &y& Elsevier]

Polyetheretherketone (PEEK) is a potential substitute for conventional metallic biomedical implants owing to its superior mechanical and chemical properties, as well as biocompatibility. However, its inherent bio-inertness and poor osseointegration limit its use in clinical applications. Herein, thin titanium films were deposited on the PEEK substrate by plasma sputtering, and porous nanonetwork structures were incorporated on the PEEK surface by alkali treatment (PEEK-TNS). Changes in the physical and chemical characteristics of the PEEK surface were analyzed to establish the interactions with cell behaviors. The osteoimmunomodulatory properties were evaluated using macrophage cells and osteoblast lineage cells. The functionalized nanostructured surface of PEEK-TNS effectively promoted initial cell adhesion and proliferation, suppressed inflammatory responses, and induced macrophages to anti-inflammatory M2 polarization. Compared with PEEK, PEEK-TNS provided a more beneficial osteoimmune environment, including increased levels of osteogenic, angiogenic, and fibrogenic gene expression, and balanced osteoclast activities. Furthermore, the crosstalk between macrophages and osteoblast cells showed that PEEK-TNS could provide favorable osteoimmunodulatory environment for bone regeneration. PEEK-TNS exhibited high osteogenic activity, as indicated by alkaline phosphatase activity, osteogenic factor production, and the osteogenesis/osteoclastogenesis-related gene expression of osteoblasts. The study establishes that the fabrication of titanate nanonetwork structures on PEEK surfaces could extract an adequate immune response and favorable osteogenesis for functional bone regeneration. Furthermore, it indicates the potential of PEEK-TNS in implant applications. [ABSTRACT FROM AUTHOR]

Various stresses latently induce cellular senescence that occasionally deteriorates the functioning of surrounding tissues. Nevertheless, little is known about the appearance and function of senescent cells, caused by the implantation of beta-tricalcium phosphate (β-TCP)—used widely in dentistry and orthopedics for treating bone diseases. In this study, two varying sizes of β-TCP granules (<300 μm and 300–500 μm) were implanted, and using histological and immunofluorescent staining, appearances of senescent-like cells in critical-sized bone defects in the calvaria of Sprague Dawley rats were evaluated. Parallelly, bone formation in defects was investigated with or without the oral administration of senolytics (a cocktail of dasatinib and quercetin). A week after the implantation, the number of senescence-associated beta-galactosidase, p21-, p19-, and tartrate-resistant acid phosphatase-positive cells increased and then decreased upon administrating senolytics. This administration of senolytics also attenuated 4-hydroxy-2-nonenal staining, representing reactive oxygen species. Combining senolytic administration with β-TCP implantation significantly enhanced the bone formation in defects as revealed by micro-computed tomography analysis and hematoxylin-eosin staining. This study demonstrates that β-TCP granules latently induce senescent-like cells, and senolytic administration may improve the bone-forming ability of β-TCP by inhibiting senescence-associated mechanisms. [ABSTRACT FROM AUTHOR]

Scaffolds stimulate cell proliferation and differentiation and play major roles in providing growth and nutrition factors in the repair of bone defects. We used the recombinant peptide Cellnest™ to prepare the three-dimensional stem cell complex, CellSaic, and evaluated whether CellSaic containing rat dental pulp stem cells (rDPSCs) was better than that containing rat bone marrow stem cells (rBMSCs). rDPSC-CellSaic or rBMSC-CellSaic, cultured with or without osteogenic induction medium, formed the experimental and control groups, respectively. Osteoblast differentiation was evaluated in vitro and transplanted into a rat model with a congenital jaw fracture. Specimens were collected and evaluated by microradiology and histological analysis. In the experimental group, the amount of calcium deposits, expression levels of bone-related genes (RUNX2, ALP, BSP, and COL1), and volume of mineralized tissue, were significantly higher than those in the control group (p < 0.05). Both differentiated and undifferentiated rDPSC-CellSaic and only the differentiated rBMSC-CellSaic could induce the formation of new bone tissue. Overall, rBMSC-CellSaic and rDPSC-CellSaic made with Cellnest™ as a scaffold, provide excellent support for promoting bone regeneration in rat mandibular congenital defects. Additionally, rDPSC-CellSaic seems a better source for craniofacial bone defect repair than rBMSC-CellSaic, suggesting the possibility of using DPSCs in bone tissue regenerative therapy. [ABSTRACT FROM AUTHOR]

Bone regeneration using mesenchymal stem cells has several limitations. We investigated adipose-derived dedifferentiated fat (DFAT) cells as an alternative, and evaluated their cell proliferation rate, osteoblast differentiation, and bone regeneration ability in combination with activated platelet-rich plasma (aPRP). Rat DFATs and aPRP were isolated using ceiling culture and centrifugation, respectively. The cell proliferation rate was measured, and the cells were cultured in an osteoblast differentiation medium under varying concentrations of aPRP for 21 days and stained with Alizarin red. Gene expression was evaluated using real time polymerase chain reaction. Critical defects were implanted with DFAT seeded gelatin sponges under aPRP, and four weeks later, the bone regeneration ability was evaluated using micro-computed tomography and hematoxylin-eosin staining. The cell proliferation rate was significantly increased by the addition of aPRP. Alizarin red staining was positive 21 days after the start of induction, with significantly higher Runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) expression levels than those in the controls. A 9 mm critical defect was largely closed (60.6%) after four weeks of gelatin sponge implantation with DFAT and aPRP. Therefore, materials combining DFAT cells and aPRP may be an effective approach for bone regeneration. Further research is needed to explore the long-term effects of these materials. [ABSTRACT FROM AUTHOR]

Despite advances in bone regenerative medicine, the relationship between stress-induced premature senescence (SIPS) in cells and bone regeneration remains largely unknown. Herein, we demonstrated that the implantation of a lipopolysaccharide (LPS) sustained-release gelatin sponge (LS-G) increases the number of SIPS cells and that the elimination of these cells promotes bone formation in critical-sized bone defects in the rat calvaria. Histological (hematoxylin–eosin and SA-β-gal) and immunohistological (p16 and p21 for analyzing cellular senescence and 4-HNE for oxidation) staining was used to identify SIPS cells and elucidate the underlying mechanism. Bone formation in defects were analyzed using microcomputed tomography, one and four weeks after surgery. Parallel to LS-G implantation, local epigallocatechin gallate (EGCG) administration, and systemic senolytic (dasatinib and quercetin: D+Q) administration were used to eliminate SIPS cells. After LS-G implantation, SA-β-gal-, p16-, and p21-positive cells (SIPS cells) accumulated in the defects. However, treatment with LS-G+EGCG and LS-G+D+Q resulted in lower numbers of SIPS cells than that with LS-G in the defects, resulting in an augmentation of newly formed bone. We demonstrated that SIPS cells induced by sustained stimulation by LPS may play a deleterious role in bone formation. Controlling these cell numbers is a promising strategy to increase bone regeneration. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide (LPS) is a well-known strong inducer of inflammation. However, there is little information regarding how LPS-release behavior affects cellular senescence at the affected area. In this paper, we demonstrate that a vacuum-heating technique (dehydrothermal treatment) can be utilized to prepare an LPS sustained-release gelatin sponge (LS-G). LPS sustained release from gelatin leads to the long-term existence of senescent cells in critical-sized bone defects in rat calvaria. Three types of gelatin sponges were prepared in this study: a medical-grade gelatin sponge with extremely low LPS levels (MG), LS-G, and a LPS rapid-release gelatin sponge (LR-G). Histological (H-E) and immunohistochemical (COX-2, p16, and p21) staining were utilized to evaluate inflammatory reactions and cellular senescence one to three weeks after surgery. Soft X-ray imaging was utilized to estimate new bone formation in the defects. The LR-G led to stronger swelling and COX-2 expression in defects compared to the MG and LS-G at 1 week. Despite a small inflammatory reaction, LS-G implantation led to the long-term existence of senescent cells and hampered bone formation compared to the MG and LR-G. These results suggest that vacuum heating is a viable technique for preparing different types of materials for releasing bacterial components, which is helpful for developing disease models for elucidating cellular senescence and bone regeneration. [ABSTRACT FROM AUTHOR]