Mimicked Periosteum Layer Based on Deposited Particle Silk Fibroin Membrane for Osteogenesis and Guided Bone Regeneration in Alveolar Cleft Surgery: Formation and in Vitro Testing.

An alveolar cleft is a critical tissue defect often treated with surgery. In this research, the mimicked periosteum layer based on deposited silk fibroin membrane was fabricated for guided bone regeneration in alveolar cleft surgery. The deposited silk fibroin particle membranes were fabricated by spray-drying with different concentrations of silk fibroin (v/v): 0.5% silk fibroin (0.5% SFM), 1% silk fibroin (1% SFM), 2% silk fibroin (2% SFM), and 1% silk fibroin film (1% SFF) as the control. The membranes were then characterized and the molecular organization, structure, and morphology were observed with FT-IR, DSC, and SEM. Their physical properties, mechanical properties, swelling, and degradation were tested. The membranes were cultured with osteoblast cells and their biological performance, cell viability and proliferation, total protein, ALP activity, and calcium deposition were evaluated. The results demonstrated that the membranes showed molecular transformation of random coils to beta sheets and stable structures. The membranes had a porous layer. Furthermore, they had more stress and strain, swelling, and degradation than the film. They had more unique cell viability and proliferation, total protein, ALP activity, calcium deposition than the film. The results of the study indicated that 1% SFM is promising for guided bone regeneration for alveolar cleft surgery.

Alteration of circRNA and lncRNA expression profile in exosomes derived from periodontal ligament stem cells undergoing osteogenic differentiation.

OBJECTIVE: This study investigated circRNA and lncRNA expression profile in exosomes derived from periodontal ligament stem cell (PDLSC) before and after its osteogenic differentiation. DESIGN: Exosomes derived from PDLSCs before (EX0) and after osteogenic induction for 5 (EX5) and 7 (EX7) days were harvested and exosomal circRNAs and lncRNAs were analyzed by RNA sequencing. Certain RNAs showing significantly altered expression were selected for qRT-PCR verification. The circRNA-miRNA-mRNA network and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed. RESULTS: All groups of exosomes showed typical characteristics under nanoparticle tracking analysis, flow cytometry assay and transmission electron microscopy. 69-557 circRNAs and 2907-11581 lncRNAs were found in EX0, EX5 and EX7, which were broadly distributed across the 24 pairs of human chromosomes. Compared with EX0, 3 circRNAs and 2 lncRNAs were up-regulated and 39 circRNAs and 5 lncRNAs down-regulated consistently through out of EX5 and EX7, p < 0.05. qRT-PCR confirmed certain those consistently expressed RNAs, such as circ lysophosphatidic acid receptor 1 (LPAR1). KEGG analysis showed that those consistent expressed RNAs closely related to TGF-beta pathway, MAPK pathway, mTOR pathway and FoxO signaling pathways regulating pluripotency of stem cells. CONCLUSIONS: Exosomal circRNAs and lncRNAs had significant expression changes during the early phase of osteogenic differentiation of PDLSCs. Further study would be taken for understanding the roles of exosomal circRNAs and lncRNAs playing in osteogenic differentiation of PDLSCs.

Exosomal circLPAR1 Promoted Osteogenic Differentiation of Homotypic Dental Pulp Stem Cells by Competitively Binding to hsa-miR-31.

Human dental pulp stem cells (DPSCs) hold great promise in bone regeneration. However, the exact mechanism of osteogenic differentiation of DPSCs remains unknown, especially the role of exosomes played in. The DPSCs were cultured and received osteogenic induction; then, exosomes from osteogenic-induced DPSCs (OI-DPSC-Ex) at different time intervals were isolated and sequenced for circular RNA (circRNA) expression profiles. Gradually, increased circular lysophosphatidic acid receptor 1 (circLPAR1) expression was found in the OI-DPSC-Ex coincidentally with the degree of osteogenic differentiation. Meanwhile, results from osteogenic differentiation examinations showed that the OI-DPSC-Ex had osteogenic effect on the recipient homotypic DPSCs. To investigate the mechanism of exosomal circLPAR1 on osteogenic differentiation, we verified that circLPAR1 could competently bind to hsa-miR-31, by eliminating the inhibitory effect of hsa-miR-31 on osteogenesis, therefore promoting osteogenic differentiation of the recipient homotypic DPSCs. Our study showed that exosomal circRNA played an important role in osteogenic differentiation of DPSCs and provided a novel way of utilization of exosomes for the treatment of bone deficiencies.

Effect of expanded bone marrow-derived osteoprogenitor cells seeded into polycaprolactone/tricalcium phosphate scaffolds in new bone regeneration of rabbit mandibular defects.

The purpose of this study was to assess and evaluate new bone formation in rabbit marginal mandibular defects using expanded bone marrow-derived osteoprogenitor cells seeded in three-dimensional scaffolds of polycaprolactone/tricalcium phosphate (PCL/TCP). Bone marrow was harvested from the rabbit ilium and rabbit bone marrow-derived osteoprogenitor cells were isolated and expanded in standard culture medium and osteogenic medium supplement. The cells were then seeded into the PCL/TCP scaffolds and the cell/scaffold constructions were implanted into prepared defects in rabbit mandibles. PCL/TCP scaffold alone and autogenous bone graft from the mandible were also implanted into the other prepared defects. The specimens were evaluated at 4 and 8 weeks after the implantation using clinical, radiographic, and histological techniques. The results of the experimental group demonstrated more newly formed bone on the surface and in the pores of the PCL/TCP scaffolds. In addition, the osteoblasts, osteocytes, and new bone trabeculae were identified throughout the defects that were implanted with the cell/scaffold constructions. The PCL/TCP alone group was filled mostly with fibrous cells particularly in the middle region with less bone formation. These results would suggest that the derived osteotoprogenitor cells have the potential to form bone tissue when seeded onto PCL/TCP scaffolds.

In vivo evaluation of modified silk fibroin scaffolds with a mimicked microenvironment of fibronectin/decellularized pulp tissue for maxillofacial surgery.

This study aimed to carry out in vivo testing of the formation of new bone by modified silk fibroin scaffolds with a mimicked microenvironment of fibronectin/decellularized pulp in bone defects. Silk fibroin scaffolds were fabricated into three-dimensional scaffolds before being coated with fibronectin/decellularized pulp. The coated scaffolds were implanted into rabbits. Twenty-four bicortical calvarial defects in 12 rabbits were divided randomly into two groups: non-coated and coated silk fibroin scaffolds. The rabbits were sacrificed 2, 4 and 8 weeks after operation for evaluation of new bone formation. The morphology of the scaffolds, new bone formation and histology were evaluated by scanning electron microscopy, micro-CT and hematoxylin and eosin staining, respectively. The results showed that the coated silk fibroin scaffolds had a fibrillar network and crystal particles in the porous structure. The coated silk fibroin scaffolds demonstrated the ability to induce the formation of new bone with low inflammation and high vascularization. The results indicated that the modified silk fibroin scaffolds showed suitable biological performance and promise for bone regeneration in maxillofacial surgery.

Assessment of bone regeneration of a tissue-engineered bone complex using human dental pulp stem cells/poly(ε-caprolactone)-biphasic calcium phosphate scaffold constructs in rabbit calvarial defects.

The objective of the present study was to investigate the effect of a fabricated combination of poly-ɛ-caprolactone (PCL)-biphasic calcium phosphate (BCP) with the modified melt stretching and multilayer deposition (mMSMD) technique on human dental pulp stem cell (hDPSC) differentiation to be osteogenic like cells for bone regeneration of calvarial defects in rabbit models. hDPSCs extracted from human third molars were seeded onto mMSMD PCL-BCP scaffolds and the osteogenic gene expression was tested prior to implantation in vivo. Two standardized 11 mm in diameter circular calvarial defects were created in 18 adult male New Zealand white rabbits. The rabbits were divided into 4 groups: (1) hDPSCs seeded in mMSMD PCL-BCP scaffolds; (2) mMSMD PCL-BCP scaffolds alone, (3) empty defects and (4) autogenous bone (n = 3 site/time point/groups). After two, four and eight weeks after the operation, the specimens were harvested for micro-CT including histological and histomorphometric analysis. The explicit results presented an interesting view of the bioengineered constructs of hDPSCs in PCL-BCP scaffolds that increased the newly formed bone compared to the empty defect and scaffold alone groups. The results demonstrated that hDPSCs combined with mMSMD PCL-BCP scaffolds may be an augmentation material for bony defect.

Biological characteristic effects of human dental pulp stem cells on poly-ε-caprolactone-biphasic calcium phosphate fabricated scaffolds using modified melt stretching and multilayer deposition.

Craniofacial bone defects such as alveolar cleft affect the esthetics and functions that need bone reconstruction. The advanced techniques of biomaterials combined with stem cells have been a challenging role for maxillofacial surgeons and scientists. PCL-coated biphasic calcium phosphate (PCL-BCP) scaffolds were created with the modified melt stretching and multilayer deposition (mMSMD) technique and merged with human dental pulp stem cells (hDPSCs) to fulfill the component of tissue engineering for bone substitution. In the present study, the objective was to test the biocompatibility and biofunctionalities that included cell proliferation, cell viability, alkaline phosphatase activity, osteocalcin, alizarin red staining for mineralization, and histological analysis. The results showed that mMSMD PCL-BCP scaffolds were suitable for hDPSCs viability since the cells attached and spread onto the scaffold. Furthermore, the constructs of induced hDPSCs and scaffolds performed ALP activity and produced osteocalcin and mineralized nodules. The results indicated that mMSMD PCL-BCP scaffolds with hDPSCs showed promise in bone regeneration for treatment of osseous defects.

Repairing calvarial defects with biodegradable polycaprolactone-chitosan scaffolds fabricated using the melt stretching and multilayer deposition technique.

The ability to repair bone defects of polycaprolactone-chitosan scaffolds containing 20% chitosan (PCL-20%CS) fabricated using the melt stretching and multilayer deposition (MSMD) technique was assessed and compared with commercial scaffolds. Two calvarium defects of 11 mm in diameter were created in each of the fifteen New Zealand white rabbits. The PCL-20%CS scaffolds were implanted in one site (group A) while another site was performed with PCL-tricalcium phosphate (TCP) scaffolds containing 20% TCP (PCL-20%TCP) fabricated by fused deposition modeling technique (FDM) (group B). At two, four and eight weeks thereafter, new bone regeneration within the defects was assessed using histomorphometric and micro-computed tomography (µ-CT) analysis. The result of histological sections demonstrated that chronic inflammatory reaction was generally detected along scaffolds of group A, but it was not found in group B. Over 8 weeks, the µ-CT analysis indicated that the average amount of new bone of group A was slightly less than that of group B (p>0.05). In conclusion, efficacy of the PCL-20%CS MSMD scaffolds for repairing bone defects was less than that of the PCL-20%TCP FDM scaffolds. However, MSMD scaffolding is still the technique of choice, but needed some modifications.

Quantitative expression of bone-related cytokines induced by mechanical tension-stress during distraction osteogenesis in a rabbit mandible.

AIM: The aim of the present study was to investigate the temporal and spatial gene expression of bone morphogenetic proteins (BMP)-2, -4, and -7, and transforming growth factor-ß (TGF-ß), during the distraction process of the rabbit mandible. METHODS: Twenty rabbits each had an osteotomy on the left mandibular body, and distraction devices were fixed. After a delay of 3 days, distraction was started at a rate of 0.5 mm/12 h for 10 days, followed by a 3-week consolidation phase. Four rabbits were killed 5 and 10 days of distraction, and 1, 2, and 3 weeks after the completion of distraction. The clinical, histological, and radiographic appearances were evaluated and analyzed with the concomitant BMP expression pattern at each interval. RESULTS: After the distraction was started, the fibrous interzone developed between the osteotomy fragments, where intramembranous ossification was noted. The quantitative expression of BMP-2, -4, and -7, and TGF-ß, were increased immediately after active distraction before a gradual decline to normal levels after the third week of consolidation. CONCLUSION: These results suggest that BMP and TGF-ß play an important role in the induction of bone formation during distraction osteogenesis. The selective expression of each bone-related cytokine could provide useful insight into accelerated bone maturation and the treatment of poorly-healing fractures in clinical cases.

Comparative study of different centrifugation protocols for a density gradient separation media in isolation of osteoprogenitors from bone marrow aspirate.

INTRODUCTION: Human bone marrow contains osteoprogenitors capable of differentiating into osteoblasts. Density gradient centrifugation (DGC) is a commonly used method to isolate osteoprogenitors from bone marrow. Numerous studies used different dilution and centrifugation protocols, which might affect cell yields and quality. Moreover, the relative isolation efficiencies of the different separation protocols have not been investigated. This study compares the enrichment efficacy of the two different centrifugation protocols for a commonly used DGC media in isolation of osteoprogenitors. MATERIAL AND METHOD: Bone marrow was aspirated from human anterior iliac crests. Osteoprogenitors are isolated with Ficoll DGC media. A centrifugal force of 400 g and 1:1 dilution was compared with the centrifugal force of 1000 g after three dilution times with a buffer. RESULTS: The average numbers of isolated cells were significantly higher when using lower centrifugal force with 1:1 dilution, however, there was no detectable difference between Colony-forming unit-fibroblast (CFU-F) forming capacity, STRO-1 positivity, osteogenic differentiation or mineralization abilities between protocols. CONCLUSION: Both protocols could isolate competent and functional osteoprogenitors, while a lower centrifugal force (400 g) with 1:1 dilution produced recovery of more osteoprogenitors.

The primacy of platelet-rich fibrin on bone regeneration of various grafts in rabbit's calvarial defects.

AIMS: This study investigated the effect of platelet-rich fibrin (PRF) on bone regeneration of various grafting materials in rabbit calvarial defects. MATERIAL AND METHODS: Two bicortical skull defects were prepared in 20 New Zealand white rabbits; 10 rabbits were treated with PRF and the other 10 were non-PRF. In both groups, autogenous bone was compare to empty defects in 5 rabbits and the composite of autogenous bone and deproteinized bovine bone versus deproteinized bovine bone (DBB) in the other five. The animals were sacrificed at 8 weeks. Bone formation was assessed by radiographic densitometry and histomorphometric analysis. RESULTS: The mean optical density (OD) and histomorphometric analysis (HA) of the percentage of new bone showed that the PRF groups were significantly higher than the non-PRF groups in the autogenous bone graft (OD: 0.60 ± 0.19 vs 0.36 ± 0.03; HA: 38.03 ± 4.23 vs 26.21 ± 10.58) and the empty defect (OD: 0.29 ± 0.06 vs 0.11 ± 0.06; HA: 18.81 ± 9.27 vs 6.24 ± 5.01), but not in the DBB group (OD: 1.18 ± 0.17 vs 1.07 ± 0.05; HA: 13.067 ± 3.64 vs 9.63 ± 5.47) and the composite group (OD: 0.81 ± 0.15 vs 0.91 ± 0.05; HA: 22.63 ± 3.61 vs 21.29 ± 3.52). CONCLUSIONS: PRF had a positive effect on bone formation when used alone or combined with autogenous bone, but not with deproteinized bovine bone.

Biomechanical properties of novel biodegradable poly ε-caprolactone-chitosan scaffolds.

AIM: To investigate the biomechanical properties of poly ε-caprolactone (PCL)-chitosan (CS) scaffolds fabricated by the melt stretching and multilayer deposition technique. METHODS: The PCL-CS scaffolds containing CS at 0% (pure PCL), 10%, and 20% by weight were prepared. For the monolayer scaffolds, shear and blending tests simulating the reconstruction of orbital floor defects (situation A) and mandibular defects (situation B) were conducted. For the 3-D scaffolds, compression tests of their superior and lateral aspects were done. RESULTS: For the monolayer scaffolds, the pure PCL group had remarkably lower shear strength than the other groups (P > 0.05). In situation A, all groups withstood the forces without any significant difference. In situation B, the pure PCL group could withstand the forces remarkably lower than those of the other group (P < 0.05). The 3-D scaffolds of all groups could withstand compressive forces directed towards their superior aspects. However, they could not withstand the forces directed towards their lateral aspects at the limited strain. CONCLUSIONS: The monolayer scaffolds were suitable for reconstruction of the orbital floor and mandibular defects under light load-bearing conditions. The 3-D scaffolds could be used in the high load bearing-areas only if the forces were directed at their superior aspects.

Biodegradable polycaprolactone-chitosan three-dimensional scaffolds fabricated by melt stretching and multilayer deposition for bone tissue engineering: assessment of the physical properties and cellular response.

Fabrication of polycaprolactone (PCL)-chitosan (CS) three-dimensional (3D) scaffolds using the novel technique of melt stretching and multilayer deposition was introduced. In brief, firstly, the PCL-CS monofilaments containing 0% (pure PCL), 10%, 20% and 30% CS by weight were fabricated by melting and stretching processes. Secondly, the desired multilayer (3D) scaffolds were fabricated by arranging and depositing the filaments. Physical properties of the filaments and the scaffolds were evaluated. MC3T3-E1 cell lines were seeded on the scaffolds to assess their proliferation. A typical micro-groove pattern was found on the surfaces of pure PCL filaments due to stretching. The filaments of PCL-30%CS had the highest tendency of fracture during stretching and could not be used to form the scaffold. Increasing CS proportions tended to reduce the micro-groove pattern, surface roughness, tensile strength and elasticity of the filaments, whilst compressive strength of the PCL-CS scaffolds was not affected. The average pore size and porosity of the scaffolds were 536.90 ± 17.91 µm and 45.99 ± 2.8% respectively. Over 60 days, degradation of the scaffolds gradually increased (p > 0.05). The more CS containing scaffolds were found to increase in water uptake, but decrease in degradation rate. During the culture period, the growth of the cells in PCL-CS groups was significantly higher than in the pure PCL group (p < 0.05). On culture-day 21, the growth in the PCL-20%CS group was significantly higher than the other groups (p < 0.05). In conclusion, the PCL-20%CS scaffolds obtained the optimum results in terms of physical properties and cellular response.

Distraction osteogenesis treatment of maxillary deficiency for cleft patient using internal distraction device: a case report.

A case of severe maxillary hypoplasia in a 21 years old male Thai patient with a complete unilateral cleft of primary and secondary palates treated by internal distraction osteogenesis for maxillary advancement is presented. Initial evaluation showed Class III malocclusion with total crossbite and Class III skeletal malrelationship. Two intraoral distractors were placed following a Le Fort I osteotomy. A maxillary advancement of 8 mms was obtained with 1 mm overjet. Following distraction, Class III elastics were used to increase the overjet until an overjet of 3 mms was obtained. Both acceptable skeletal and soft tissue relationships and satisfactory occlusion have been produced. After 20 months of postoperative follow-up, the occlusal result is stable and skeletal relapse can not be detected.

Distraction osteogenesis: role and clinical applications in the maxillofacial region.

Distraction osteogenesis or callostasis is a technique of new bone formation by gradual separation of bony fragments. The method was first developed for limb lengthening but recently this process was widely applied to the cranio-maxillofacial bones. The application included unlimited bone lengthening and reconstruction of segmental defects. Several designs of extra-oral and intra-oral distraction devices were invented to suit different areas of craniofacial bone. Nevertheless intraoral distractors have several advantages including minimal scarring and being less cumbersome. Clinical cases using distraction osteogenesis as an alternative treatment to conventional surgical procedures for maxillo-mandibular lengthening and reconstruction of alveolar segmental defects after tumour resection before implant installation are presented and discussed.

Osteoconductive effects of 3 heat-treated hydroxyapatites in rabbit calvarial defects.

PURPOSE: This study aimed to detect the osteoconductive ability of 3 bovine hydroxyapatites (HAs) that were sintered at 800 degrees C (HA800), 1,200 degrees C (HA1200), and 1,350 degrees C (HA1350), according to new bone formation. MATERIAL AND METHODS: Two bicortical skull defects were prepared in 10 New Zealand white rabbits. Four rabbits were used as controls; in each, 1 defect was filled with autogenous bone chips and the contralateral defect was left empty for the critical size defect (CSD). The other 6 rabbits had a total of 12 defects, 4 each randomly filled with HA 800, HA1200, or HA1350. The animals were sacrificed at 8 weeks. New bone formation was assessed by radiographic densitometry and histomorphometric analysis. RESULTS: The mean optical density (OD) of the CSD group (0.092 +/- 0.006) was less than that of the autogenous bone chip (0.102 +/- 0.002), HA1200 (0.108 +/- 0.005), and HA 1350 (0.102 +/- 0.003) groups. The mean OD of the HA 1200 group was significantly different from that of the HA 800 group (0.094 +/- 0.003). The histomorphometric analysis demonstrated that the autogenous bone chip group (34.89 +/- 4.61) was significantly different from the CSD (12.16+/- 6.97), HA 800 (18.32 +/- 7.33), and HA1350 (13.99 +/- 3.94) groups, but not the HA1200 group (24.83 +/- 12.12). CONCLUSIONS: The findings demonstrate that heat-treated bovine HA enhances bone formation, and HA 1200 tends to provide greater bone formation than the other 2 HAs.

Two uncommon uses of Bio-Oss for GTR and ridge augmentation following extractions: two case reports.

Bio-Oss is natural bovine bone mineral, which has the property of bone conduction. It is recommended to be used in two- or three-walled bony defects with an ample supply of pleuripotential cells. Two cases are reported. The first was an intentional replantation, because of previous trauma, of a hopeless tooth affected with severe periodontitis. The tooth was replanted after complete elimination of granulation tissue. Bio-Oss, together with a guided tissue regeneration (GTR) membrane, was used to enhance periodontal regeneration. After 2 years of follow-up, the replanted tooth was quite stable. In the second case, Bio-Oss, together with bone taken from the retromolar area, was used in a sinus lift grafting procedure after the removal of two supernumerary teeth from the floor of the maxillary sinus. Four months after grafting, an orthodontic treatment was applied to move the two adjacent teeth through the grafted site and align them in the proper position. The clinical results of the two cases were satisfactory.