Prisana Pripatnanont

Properties of chitosan–collagen sponges and osteogenic differentiation of rat-bone-marrow stromal cells

The aim of this study was to further investigate effects of a combined chitosan and collagen matrix on osteogenic differentiation of rat-bone-marrow stromal cells (BMSCs), including analysis of the physical and mechanical properties of the sponges. There were 4 study groups: collagen, chitosan, 1:1 chitosan-collagen and 1:2 chitosan-collagen sponges. Chitosan-collagen sponges were fabricated using the freeze-drying technique. BMSCs were seeded on the sponges and cultivated in mineralized culture medium for 27 days. Attachment and growth of cells on the sponges were examined under a scanning electron microscope. Alkaline phosphatase activity and levels of osteocalcin were monitored. Tests of swelling, collagenase and lysozyme enzymatic degradation, and mechanical strength were performed. The BMSCs attached successfully to the structure of the sponges, and expression of ALP and osteocalcin on collagen and chitosan-collagen composite sponges was greater than on chitosan sponges. All sponges showed a high degree of water uptake. Chitosan and chitosan-collagen sponges showed a higher resistance to enzymatic degradation than collagen sponges. A 1:1 chitosan-collagen sponge demonstrated the highest compressive strength. Combined chitosan-collagen matrixes promoted osteoblastic differentiation of BMSCs, and improved the mechanical and physical properties of the sponges.

Fabrication and characterization of novel nano hydroxyapatite/β-tricalcium phosphate scaffolds in three different composition ratios†

The biphasic calcium phosphate (BCP) concept was introduced to overcome disadvantages of single phase biomaterials. Different composition ratios of BCP bioceramics have been studied, yet controversies regarding the effects of ratio on biomaterial behavior still exist. In this study, BCP scaffolds were prepared from nano hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) that were synthesized via a solid state reaction. Three different composition ratios of pure BCP and collagen-based BCP scaffolds (%HA/%β-TCP; 30/70, 40/60, and 50/50) were produced using a polymeric sponge method. Physical and mechanical properties of all materials and scaffolds were investigated. SEM showed overall distribution of both macropores (80-200 μm) and micropores (0.5-2 μm) with high interconnected porosities. Total porosity of pure BCP (90% ± 3%) was found to be higher than collagen-based BCP (85% ± 2%). It was observed that following sintering process, dimensional shrinkage of large scaffolds (39% ± 4%) was lower than small ones (42% ± 5%) and scaffolds with high HA ratios (50%) experienced higher dimensional changes than those with higher β-TCP (70%) ratios (45% ± 3% and 36% ± 1%, respectively). Compressive strength of both groups was less than 0.1 MPa and collagen coating had almost no influence on mechanical behavior. Further studies may improve the physical properties of these scaffolds and investigate their exact biological behaviors.

Proportion of deproteinized bovine bone and autogenous bone affects bone formation in the treatment of calvarial defects in rabbits

This study assessed new bone formation generated using three different proportions of autogenous bone (AB) and deproteinized bovine bone (DBB). Thirty bicortical skull defects were prepared in 15 rabbits, divided into 3 groups: Group 1, critical size defect (CSD) versus AB as controls; Group 2, DBB versus a composite of AB and DBB using a proportion of 1:1; and Group 3, a composite of AB and DBB using a proportion of 1:2 versus a proportion of 1:4. After 8 weeks, radiographic evaluation was assessed using densitometry and new bone formation by histomorphometry. The mean optical density of the CSD (0.108+/-0.238) and AB (0.352+/-0.161) groups differed significantly from the DBB group (1.044+/-0.093) and the groups using a proportion of 1:1 (0.905+/-0.078), 1:2 (0.865+/-0.294) and 1:4 (0.867+/-0.304). Histomorphometry revealed a higher percentage of new bone in the AB group (30.223+/-16.722) than in the groups using proportions of 1:2 (22.639+/-5.659), 1:1 (20.929+/-6.169), 1:4 (9.621+/-2.400), DBB (14.441+/-2.742) and CSD (10.645+/-8.868), respectively. The 1:2 group had significantly higher bone content than the 1:4 group. The proportions of 1:1 and 1:2 resulted in greater bone formation than the proportion of 1:4, DBB and CSD.

A comparison of autogenous bone graft combined with deproteinized bovine bone and autogenous bone graft alone for treatment of alveolar cleft.

This study assessed the use of composite autogenous bone and deproteinized bovine bone (DBB) for repairing alveolar cleft compared with autogenous bone alone in terms of clinical outcomes and patient morbidity. 30 patients with a mean age of 10.2±1.7 years were randomly divided into two groups. Group I used autogenous cancellous bone graft harvested from the anterior iliac crests by the conventional trapdoor approach. Group II used a composite of DBB and autogenous cancellous bone harvested by a trephine bone collector; the proportion of 1:1 by volume was used. The bone graft quantities of both groups decreased with time. Their average changes were not statistically different over 24 months after grafting. The canines of both groups could spontaneously or orthodontically erupt through the grafting areas. Patients in group II recovered from uncomfortable walking significantly faster than those in group I (p<0.05) and their duration of hospital stay was significantly shorter than those in group I (p<0.05). The average operation time, intra-operative blood loss and postoperative pain were less in group II than in group I (p>0.05).

In vitro biocompatibility analysis of novel nano-biphasic calcium phosphate scaffolds in different composition ratios.

This study aimed to evaluate in vitro biocompatibility of a composite of nanoscale biphasic calcium phosphate (BCP) and collagen (C) compared to pure BCP (P) in different composition ratios of nanohydroxyapatite to nano-β-tricalcium phosphate (HA/β-TCP). Each study group comprised of three ratios of BCP (30/70, 40/60, and 50/50). For evaluation of cellular response toward each ratio, mouse osteoblast (MC3T3-E1) cell line was cultivated on the scaffolds for 19 days. Analysis of cell proliferation, cell viability, cell attachment and morphology, alkaline phosphatase (ALP) activity, and osteocalcin synthesis were done on culture days 1, 3, 7, 13, 15, and 19, appropriately. The scanning electron microscopy showed that the osteoblasts attached successfully to scaffolds surfaces in both BCP groups and in all different ratios by spreading their filopodia and expressing similar viability that was confirmed by confocal laser scanning electron microscope. BCP scaffold (P3070) showed remarkable ALP activity, whereas BCP (P5050) showed highest osteocalcin activity. Collagen coating supported high cell proliferation on culture day 1 and possessed limited benefit restricted to early phase of cell differentiation. In conclusion, the fabricated nanoscale BCP scaffolds offered high biocompatibility and supported well the cell proliferation and differentiation regardless the composition ratio. Furthermore, higher ratio of TCP supported the early phase of cell proliferation, whereas higher HA ratio influenced the later phase. Finally, BCP scaffolds P5050 and C4060 were suggested as candidates for clinical applications.

Bone Regeneration Potential of Biphasic Nanocalcium Phosphate with High Hydroxyapatite/Tricalcium Phosphate Ratios in Rabbit Calvarial Defects.

PURPOSE This study aimed to evaluate the effect of biphasic calcium phosphate (BCP) with high hydroxyapatite/tricalcium phosphate (HA/TCP) ratios on bone formation in rabbit calvarial defects. MATERIALS AND METHODS Sixteen New Zealand white rabbits were randomly divided into two groups, a control group and an experimental group. In each animal, bilateral circular defects (10-mm diameter) were created on the calvarium. In the control group (three rabbits per time frame), defects were grafted with autogenous bone chips in one side and left empty in the other side. In the experimental group (five rabbits per time frame), defects were grafted with BCP1 (HA:TCP, 8:2) in one side and BCP2 (HA:TCP, 9:1) in the contralateral side. The animals were sacrificed at 2 and 8 weeks as designated. Bone formation and residual grafting material were assessed by radiographic densitometry, microcomputed tomography (micro-CT), and histomorphometric analysis. RESULTS Histologic observation revealed that BCP1, BCP2, and the autogenous bone group preserved good contours of the defect, while the unfilled defect group showed connective tissue healing. Micro-CT analysis at 8 weeks showed the comparable percentages of bone volume fraction (% BV/TV) of BCP1 (20.70% ± 2.76%) and BCP2 (20.72% ± 3.97%) and two times higher than that of 2 weeks (9.90% ± 0.75%, 10.57% ± 0.85%). The autogenous group had a significantly (P < .005) greater % BV/TV (34.58% ± 8.85%) than other groups. The percentage of the material volume fraction of BCP1 and BCP2 was not different. The histomorphometry demonstrated a higher increase in newly formed bone from 2 to 8 weeks in all groups, and all were comparable (autogenous: 4.30% ± 0.76%, 12.83% ± 7.74%; unfilled: 2.82% ± 1.19%, 8.14% ± 6.35%; BCP1: 3.01% ± 2.57%, 8.81% ± 3.86%; BCP2: 3.24% ± 1.09%, 10.27% ± 3.98%). CONCLUSION BCP with a high ratio of HA presented good osteoconductive properties and space-maintaining capacity and would be beneficial for long-term preservation or when stable graft volume is essential.

Effect of osteogenic periosteal distraction by a modified Hyrax device with and without platelet-rich fibrin on bone formation in a rabbit model: A pilot study

This study evaluated the effect of a modified Hyrax device and platelet-rich fibrin (PRF) on osteogenic periosteal distraction (OPD). Twelve adult male New Zealand white rabbits were separated into two main groups (six in each) according to the duration of the consolidation period (4 or 8 weeks). In each main group, the animals underwent OPD of the left and right sides of the mandible and were divided into four subgroups (three animals per group): device vs. device+PRF, and PRF vs. sham. Radiographic, histological, histomorphometric, and micro-computed tomography (micro-CT) analyses were performed. New bone formation was observed on the lateral and vertical sides of the mandible of all groups. Micro-CT and histomorphometry showed that the device+PRF group presented the highest percentages of bone volume and bone area at 4 weeks (56.67 ± 12.67%, 41.37 ± 7.57%) and at 8 weeks (49.67 ± 8.33%, 55.46 ± 10.67%; significantly higher than the other groups, P<0.001), followed by the device group at 4 weeks (33.00 ± 1.73%, 33.21 ± 11.00%) and at 8 weeks (30.00 ± 3.00%, 23.25 ± 5.46%). In conclusion, the modified Hyrax device was used successfully for OPD in a rabbit model to gain vertical ridge augmentation, and greater bone maturation was achieved with the addition of PRF.

Allogenic human serum, a clinical grade serum supplement for promoting human periodontal ligament stem cell expansion

Exposing human periodontal ligament stem cells (hPDLSCs) to animal proteins during cell expansion would compromise quality and safety of the hPDLSCs for clinical applications. The current study aimed to evaluate the replacement of animal‐based serum by human serum for the expansion of hPDLSCs. hPDLSCs were cultured in culture media supplemented with four types of serums: Group A: fetal bovine serum (FBS); Group B: allogeneic human male AB serum (HS); Group C: in‐house autologous (Auto‐HS); and Group D: in‐house allogeneic human serums (Allo‐HS). Exhibitions of mesenchymal stem cell characteristics of hPDLSCs were examined. Then, growth and osteogenic (OS) differentiation potential of hPDLSCs in FBS and HS at passages 5 and 15 were compared to investigate the effects of serum supplements on growth and expansion stability of the expanded hPDLSCs. After that, growth and OS differentiation of hPDLSCs in Auto‐ and Allo‐HS were investigated. Flow cytometrical analyses, functional differentiations, cell growth kinetic, cytogenetic analysis, alkaline phosphatase and calcium content assays, and oil red O and von Kossa staining were performed. Results showed that at passage 5, HS promoted growth and OS differentiation of hPDLSCs and extensive cell expansion, decreased growth and differentiation potential of the expanded hPDLSCs, particularly in HS. Growth and OS differentiation of hPDLSCs in Auto‐HS and Allo‐HS were not different. In summary, allogeneic human serum could be a replacement to FBS for hPDLSC expansion. In vitro cell expansion of hPDLSCs should be minimal to ensure optimal cell quality. Copyright

Dimensional Change and Microstructure of Intraoral Bone Block Grafts Covered with Platelet-Rich Fibrin and a Barrier Membrane in Ridge Augmentation: A Pilot Investigation

This study investigated the dimensional change and microstructure of intraoral bone block grafts covered with platelet-rich fibrin (PRF) and a barrier membrane in ridge augmentation. Seven patients were included, with 18 implant sites (10 ramus and 8 symphysis). The average final ridge width gains in the ramus from cast-based measurement (3.86 ± 0.87 mm) and cone beam computed tomography measurements (3.63 ± 1.38 mm) were not different from the symphysis (3.36 ± 2.26 mm and 3.44 ± 1.52 mm, respectively). Ridge width reduction of the ramus (-10.46 ± 10.55%) was higher than the symphysis (-5.04 ± 2.08%). The ramus showed bone volume fractions from the microcomputed tomography (84.66 ± 8.36%) and percentage of bone area from histomorphometry (80.29 ± 12.03%) that were comparable to those for the symphysis (83.13 ± 8.1% and 84.98 ± 14.50%, respectively). The dimensional change of the intraoral block graft covered with PRF and a resorbable membrane was minimal, and the symphysis graft was less resorbed than the ramus graft. Nevertheless, their microstructures were comparable.

The Fabricated Collagen-Based Nano-Hydroxyapatite/β-Tricalcium Phosphate Scaffolds

The biphasic calcium phosphate (BCP) concept was introduced to overcome disadvantages of single phase biomaterials. In this study, we prepared BCP from nanoHA and β-TCP that were synthesized via a solid state reaction. Three different ratios of pure BCP and collagen-based BCP scaffolds (%HA/%β-TCP; 30/70, 40/60 and 50/50) were produced using a polymeric sponge method. Physical and mechanical properties of all materials and scaffolds were investigated. XRD pattern proved the purity of each HA, β-TCP and BCP. SEM showed overall distribution of macropores (80-200 µm) with appropriate interconnected porosities. Total porosity of pure BCP (93% ± 2) was found to be higher than collagen-based BCP (85%± 3). It was observed that dimensional shrinkage of larger scaffold (39% ± 4) is lower than smaller one (42% ± 5) and scaffolds with higher HA (50%) ratio experienced greater shrinkage than those with higher β-TCP (70%) ratio (45% ±3 and 36% ±1 respectively). Mechanical properties of both groups tend to be very low and collagen coating had no influence on mechanical behavior. Further studies may improve the physical properties of these composite BCP.