Β-tricalcium Phosphate Blocks Research Articles

Bone regeneration using β‐tricalcium phosphate (β‐TCP) block with interconnected pores made by setting reaction of β‐TCP granules

We fabricated an interconnected dual porous β-tricalcium phosphate (β-TCP) block via a setting reaction of β-TCP granules. This β-TCP block was unique because it exhibits a fully interconnected macroporous structure with micropores in the walls surrounding macropores and a roughened surface. The porosity and diametral tensile strength of the resulting product were 58.1 ± 1.7% and 1.4 ± 0.2 MPa, respectively. Rabbit distal femur bone defects were reconstructed using the porous β-TCP block and the efficacy of the porous β-TCP block as an artificial bone substitute was evaluated histomorphometrically. For a dense β-TCP control, 4 weeks following implantation, only 0.2 ± 0.1% of the β-TCP was resorbed, and the amount of newly formed bone was limited (0.1 ± 0.1%), whereas when the defect was reconstructed with porous β-TCP, 9.2 ± 3.1% was resorbed, and the amount of new bone was 18.9 ± 5.5%. This represents an approximately 50-fold enhancement in resorption and a 200-fold increase in bone formation for our porous β-TCP block. Therefore, interconnected dual porous β-TCP made via β-TCP granule setting has good potential as an artificial bone substitute.

Feasibility evaluation of low-crystallinity β-tricalcium phosphate blocks as a bone substitute fabricated by a dissolution-precipitation reaction from α-tricalcium phosphate blocks.

Although sintered β-tricalcium phosphate blocks have been used clinically as artificial bone substitutes, the crystallinity of β-tricalcium phosphate, which might dominate biocompatibility, is extremely high. The objective of this study is to evaluate the feasibility of fabricating low-crystallinity β-tricalcium phosphate blocks, which are expected to exhibit good biocompatibility via a dissolution-precipitation reaction of α-tricalcium phosphate blocks as a precursor under hydrothermal conditions at 200°C for 24 h. Although β-tricalcium phosphate is a metastable phase, the presence of Mg2+ in the reaction solution inhibits the formation of its corresponding stable phase and induces β-tricalcium phosphate formation under acidic conditions. It was found that low-crystallinity β-tricalcium phosphate blocks could be fabricated from α-tricalcium phosphate blocks immersed in 1.0 mol/L MgCl2 + 0.1 mol/L NaH2PO4 solution while maintaining the shape of the α-tricalcium phosphate blocks. The crystallite size of the fabricated β-tricalcium phosphate blocks was 42 nm, which was substantially smaller than that of the sintered β-tricalcium phosphate blocks. When the fabricated β-tricalcium phosphate blocks were implanted into bone defects in rabbit femurs, they exhibited excellent tissue responses. In particular, the initial osteoconductivity (two and four weeks) was substantially greater than that of sintered β-tricalcium phosphate blocks.

Formation of bone in critical calvarias defects in rats under the influence of bisphosphonate alendronate Na complex

The aim of the study was to investigate dynamic of tissue structures in critical defects of calvaria of rats after inoculation of bisphosphonate (BF) alendronate Na complex into bone defects. Animal model included 24 Wistar rats divided in 3 groups: spontaneous healing under blot clot (1), inoculation of carbonate hydroxide apatite β-tricalcium phosphate blocks (2) and BF alendronate Na complex (3) on 15, 30, 60 and 90 experiment day. New bone formation was observed in group 3 as opposed to groups 1 and 2.

Ridge Preservation After Tooth Extraction With Buccal Bone Plate Deficiency Using Tunnel Structured β-Tricalcium Phosphate Blocks: A 2-Month Histologic Pilot Study in Beagle Dogs.

Reduction in alveolar ridge volume is a direct consequence of tooth extraction. Tunnel β-tricalcium phosphate (β-TCP) blocks were manufactured from randomly organized tunnel-shaped β-TCP ceramic. Efficacy of these blocks compared to extraction alone for alveolar ridge preservation after tooth extraction with buccal bone deficiency was evaluated. Maxillary first premolars of six beagle dogs were extracted after removing the buccal bone, and bone defects of 4 × 4 × 5 mm (mesio-distal width × bucco-palatal width × depth) were created. Fresh extraction sockets with buccal bone defects were filled with tunnel β-TCP blocks at test sites. Two months after the operation, histologic and histometric evaluations were performed. Regarding histologic sections, coronal and middle horizontal widths of the alveolar ridge were significantly greater at test sites (3.2 ± 0.5 and 3.6 ± 0.4 mm, respectively) than at control sites (1.2 ± 0.3 and 2.0 ± 0.6 mm, respectively). The amount of woven bone was significantly greater at test sites (62.4% ± 7.9%) than at control sites (26.8% ± 5.3%), although that of connective tissue and bone marrow was significantly greater at control sites (38.1% ± 6.2% and 16.0% ± 6.9%, respectively) than at test sites (10.7% ± 5.7% and 4.1% ± 2.2%, respectively). Regarding basic multicellular units, no statistically significant difference was found between the test and control sites (0.5% ± 0.1% and 0.6% ± 0.1%, respectively). Tunnel β-TCP blocks represent an effective bone-graft material for alveolar ridge preservation in fresh extraction sockets with buccal bone defects.

Use of β-tricalcium phosphate grafts for Airway reconstruction in rabbits: A pilot study

To assess the safety and feasibility of using β-tricalcium phosphate grafts in airway reconstruction in rabbits. Interventional animal study. Ten New Zealand White rabbits underwent division of the anterior cricoid cartilage with interposition of a graft sculpted from β-tricalcium phosphate blocks. Grafts were secured with sutures or were self-retaining. Rabbit larynges were harvested according to the following schedule: one at 1 week, one at 3 months, and two each at 2 weeks, 4 weeks, 6 months, and 1 year after graft implantation. Specimens were evaluated grossly for overall graft incorporation. Sections through the graft substance were prepared for histology. All sections were reviewed by a pathologist for evidence of vascular ingrowth, granulation tissue, inflammation, epithelialization, and graft resorption. There were no major postoperative complications. Gross examination revealed persistent widening of the airway at the level of graft placement. Histologic evaluation of the larynges showed growth of new blood vessels within all specimens and absence of significant granulation tissue near the airway lumen. The graft was at least partially covered by an epithelium in seven of the 10 specimens. Evidence of graft resorption was present as early as 4 weeks after implantation, with replacement of the graft substance by fibrous tissue, fatty tissue, bone, or cartilage. β-tricalcium phosphate grafts can be used safely in airway reconstruction procedures in rabbits. Further research is necessary to demonstrate its safety in human airway reconstruction in addition to further clarifying the processes of graft resorption and integration. NA Laryngoscope, 126:E255-E260, 2016.

Effect of Sintering Temperature on the Diametral Tensile Strength of β-Tricalcium Phosphate Block

The effect of sintering temperature on the diametral tensile strength of β-tricalcium phosphate (β-TCP) block was investigated. Uniaxially pressed block of mixture of calcium hydrogen phosphate dihydrates (CaHPO42H2O) and calcium carbonate (CaCO3) were pressed at 5 MPa and sintered at 1000°C, 1050°C and 1100°C. XRD analysis revealed that single phase β-TCP was obtained at all sintering temperature. The porosity obtained from β-TCP block sintered at 1100°C was found to be lower than that sintered at 1000°C and 1050°C. Diametral tensile strength (DTS) value of β-TCP block sintered at 1100°C showed significantly higher value than β-TCP block sintered at lower temperature. It is therefore concluded that the increased in sintering temperature affects the mechanical strength of the β-TCP block.

Construction of an osteochondral-like tissue graft combining β-tricalcium phosphate block and scaffold-free mesenchymal stem cell sheet

BackgroundAiming to construct an osteochondral-like structure, the combination of a β-tricalcium phosphate (βTCP) block with a scaffold-free sheet formed using mesenchymal stem cells (MSCs) was investigated. MethodsHuman bone marrow MSCs in a cell culture insert that was set in a 24-well plate were cultivated using a chondrogenic medium containing dexamethasone, IGF-1, and TGFβ3 for 3weeks during which a cylindrical βTCP block was put on the sheet at day 1, and the cell sheet construct was harvested. In other experiments, at day 14, the construct was put on a cell sheet that was prepared the day before and cultivated for 3weeks. ResultsThe addition of a βTCP block resulted in a combined osteochondral-like construct and comparable staining intensity by Alcian blue, while the expression levels of the aggrecan and type II collagen genes decreased a little. During the culture with the βTCP block, the expression levels of the aggrecan gene increased monotonically. The increase in the inoculum cell number from 1.86 to 3.72×106 cells resulted in marked increases in the thickness of cell sheet parts in the βTCP block and expression levels of the aggrecan and type II collagen genes, while the thickness of cell sheet parts on the βTCP block scarcely changed. On the other hand, the addition of a cell sheet that was prepared a day before to the construct at day 14 resulted in the marked increase in thickness of the cell sheet part on the βTCP block, while the thickness of that in the βTCP block did not increase. ConclusionA combined osteochondral-like structure was produced by putting a βTCP block on the sheet of MSC. The thickness of the cell sheet parts in and on the βTCP block could be increased by the increase in inoculum cell number and by providing an additional cell sheet, respectively.

Construction of osteochondral-like tissue graft combining β-tricalcium phosphate block and scaffold-free centrifuged chondrocyte cell sheet

BackgroundThe combination of a β-tricalcium phosphate (βTCP) block with a scaffold-free chondrocyte sheet formed by the centrifugation of chondrocytes in a well was investigated with the aim of constructing an osteochondral-like structure. MethodsHuman and porcine articular cartilage chondrocytes were respectively centrifuged in a 96-well plate or cell culture insert (0.32cm2) that was set in a 24-well plate, cultivated in the respective vessel for 3 weeks, and the cell sheets were harvested. In some cases, a cylindrical βTCP block (diameter 5 mm, height 3 mm) was placed on the sheet on days 1–7. The sheet size, cell number, and sulfated glycosaminoglycan accumulation were determined. ResultsThe use of a 96-well plate for not suspension but adhesion culture and the initial centrifugation of a well containing cells were crucial to obtaining a uniformly thick cell sheet. The glycosaminoglycan density of the harvested cell sheet was comparable to that of the pellet culture. An inoculum cell number of more than 31 × 105 cells tended to result in a curved cell sheet. Culture involving 18.6 × 105 cells and the 96-well plate for adhesion culture showed no curving of the cell sheet (thickness of 0.85 mm), and these were found to be the best of the culture conditions tested. The timing of the addition of a βTCP block to the cell sheet (1–7 days) markedly affected the balance between the thickness of cell sheet parts on and in the βTCP block. ConclusionCentrifugation and subsequent cultivation of chondrocytes (18.6 × 105 cells) in a 96-well plate for adhesion culture led to the production of a scaffold-free cartilage-like cell sheet with a thickness of 0.85mm. A combined osteochondral-like structure was produced by putting a βTCP block on the cell sheet. The thickness of the cell sheet on the βTCP block and the binding strength between the cell sheet and the βTCP block could be optimized by adjusting the inoculum cell number and timing of βTCP block addition.

Histological and histomorphometrical comparative study of β-tricalcium phosphate block grafts and periosteal expansion osteogenesis for alveolar bone augmentation

This study evaluated local tissue reaction around the β-tricalcium phosphate (β-TCP) block and compared results with β-TCP block grafting and periosteal expansion osteogenesis (PEO). The mandibular premolars were extracted from five dogs and buccal corticotomy was performed. Narrow alveolar ridge models were created at 4 weeks. The β-TCP block graft, such as veneer graft, was used on the right side and PEO using β-TCP block on the left side. Changes of alveolar width, histological findings and histomorphometrical analysis were evaluated. There were no problems with materials at any of the sites at any time. In both groups, the width increased after surgery and results were stable 8 weeks after surgery. Newly formed bone tissue was observed inside the β-TCP block in both sides. Histological findings differed especially at the division between mandibular bone and β-TCP block. Histomorphometric analyses revealed that β-TCP had been absorbed (mean decrease 28%) and new bone had formed (mean increase 43%) at 8 weeks postoperatively on both sides. The β-TCP block worked as a space-maker under the soft tissue, including the periosteum, and acted as a substitute for original bone. This bone substitute was effective material for bone augmentation in both methods.

Implant placement for periosteal expansion osteogenesis using β-tricalcium phosphate block: An experimental study in dogs

This study was performed to evaluate the clinical outcome of periosteal expansion osteogenesis for correction of a horizontally deficient alveolar ridge, stability of dental implants placed in the expanded areas, and osteocompatibility of beta-tricalcium phosphate (beta-TCP) block areas. The mandibular premolars were extracted and buccal corticotomy was performed in 5 female dogs. Narrow alveolar ridge models were produced at 10 weeks. The beta-TCP block was placed at the lateral surface of the mandibular bone and 2 titanium screws were inserted from the lingual aspect to push the block to the buccal side. After a latency period of 8 days, during which time primary wound healing occurred, the lingual screws were advanced by approximately 0.5 mm/day for 6 days. The expansion areas were left untreated for 8 weeks. Then implants (diameter 3.5 mm, length 9 mm) were inserted into the gap between the beta-TCP block and the original alveolar bone. We evaluated the changes in alveolar width, resonance frequency analysis of implants, and histomorphometric analysis of the beta-TCP block. No problems with the materials were observed at any of the sites of intervention before, during, or at the end of the experimental period. The width increased after expansion and showed stable results on week 8 from the end of expansion. Implants were placed in the expansion area and showed sufficient stability with slight increases in the implant stability quotient value until 8 weeks after implant placement. The amount of remaining beta-TCP decreased significantly compared with the original amount of material inserted. The mean values remaining inside the block were 44.6 +/- 8.2% and 32.1 +/- 12.0% at 8 and 16 weeks of consolidation, respectively, whereas newly formed bone comprised 20.2 +/- 7.2% and 33.5 +/- 9.5%, respectively. Newly formed bone could be acquired by periosteal expansion osteogenesis using a beta-TCP block for implant placement in a dog model. However, the bone volume was not stable after implant placement despite sufficient implant stability for 8 weeks.

A three-dimensional cell-loading system using autologous plasma loaded into a porous β-tricalcium-phosphate block promotes bone formation at extraskeletal sites in rats

The effects of platelet-rich plasma (PRP) and platelet-poor plasma (PPP) on bone marrow stromal cells (MSCs) with respect to proliferation, osteogenic differentiation, and bone formation capability were investigated. MSCs derived from rats were cultured in medium containing mixtures of PRP and PPP. Fibrinogen was eliminated prior to the experiment. The DNA content and alkaline phosphatase (ALP) activity were measured. PRP stimulated cell proliferation and inhibited osteoblastic differentiation. To examine the effects of fibrin in plasma, MSCs were cultured in PRP or PPP fibrin gels formed both on a cell culture insert installed in a culture well and on the bottom surface of the same culture well. The ALP activities of the MSCs in both of the gels were higher than those on the surface of the culture wells. The MSCs cultured on the PPP gel showed the highest ALP activity. The effects of PRP and PPP used as scaffolds for bone formation were also investigated. MSCs were suspended in PRP or PPP, introduced into porous β-tricalcium phosphate blocks, and then implanted into subcutaneous sites. Subsequently, bone formation was quantified. Further in vivo studies found that implants prepared using PPP had a greater osteoinductive capability than implants prepared with PRP.

Platelet-rich plasma improves expansion of human mesenchymal stem cells and retains differentiation capacity and in vivo bone formation in calcium phosphate ceramics

Introduction: Mesenchymal stem cells (MSC) applied to bone substitution materials can improve bone healing. Bone formation in biocomposites is highly dependent on the kind of biomaterial, its pre-treatment and the applied cells. Potentially immunogenic or infectious supplements such as fetal calf serum (FCS) should be avoided in cell expansion media. Therefore, we developed an expansion protocol free of xenogenic supplements. Cells expanded with two different media were tested on distinct biomaterials for their bone formation capacity after ectopic implantation in vivo, as well as for their growth rate and differentiation capacity in vitro.Methods: MSC of six donors were expanded with cell expansion medium containing FCS (2%) or platelet-rich plasma (PRP, 3%). Their growth rate and osteogenic, adipogenic and chondrogenic differentiation capacity were compared in vitro. For the in vivo bone formation assay, expanded cells (2 × 105 or 2 × 106) were seeded on calcium-deficient hydroxyapatite (CDHA; n = 12) and on β-tricalcium phosphate (β-TCP; n = 12) blocks, which had been coated with either fibronectin or human serum. They were then implanted subcutaneously in severe combined immunodeficient mice (SCID), harvested after 8 weeks and analysed by histology. Bone formation was assessed by a semi-quantitative bone score, after toluidine blue and alizarin red staining. Human cells were detected by an in situ hybridisation for human-specific alu sequences.Results: PRP-supplemented expansion medium yielded two-fold higher cell numbers compared to medium with FCS (P = 0.046) after 3 weeks (four passages) and retained a similar capacity to differentiate towards the osteogenic, chondrogenic and adipogenic lineage. In vivo bone formation was equal for cells expanded with PRP and FCS and depended on the specific surface area of the carrier. CDHA (specific surface area (SSA) 48 m2/g) showed a significantly better bone formation in deep layers (P = 0.005) than β-TCP (SSA 0.5 m2/g). Fibronectin-coating of the ceramics was slightly superior to coating with human serum (P = 0.045).Conclusions: The replacement of FCS by PRP eliminated risks connected with the use of xenogeneic supplements. It improved expansion of MSC and retained their differentiation and in vivo bone formation capacity in a setting adaptable to autogenous use.

The influence of mechanical strain on adult rat marrow stromal cell dynamics

The combination of a β-tricalcium phosphate (βTCP) block with a scaffold-free chondrocyte sheet formed by the centrifugation of chondrocytes in a well was investigated with the aim of constructing an osteochondral-like structure.Human and porcine articular cartilage chondrocytes were respectively centrifuged in a 96-well plate or cell culture insert (0.32 cm2) that was set in a 24-well plate, cultivated in the respective vessel for 3 weeks, and the cell sheets were harvested. In some cases, a cylindrical βTCP block (diameter 5 mm, height 3 mm) was placed on the sheet on days 1–7. The sheet size, cell number, and sulfated glycosaminoglycan accumulation were determined.The use of a 96-well plate for not suspension but adhesion culture and the initial centrifugation of a well containing cells were crucial to obtaining a uniformly thick cell sheet. The glycosaminoglycan density of the harvested cell sheet was comparable to that of the pellet culture. An inoculum cell number of more than 31 × 105 cells tended to result in a curved cell sheet. Culture involving 18.6 × 105 cells and the 96-well plate for adhesion culture showed no curving of the cell sheet (thickness of 0.85 mm), and these were found to be the best of the culture conditions tested. The timing of the addition of a βTCP block to the cell sheet (1–7 days) markedly affected the balance between the thickness of cell sheet parts on and in the βTCP block.Centrifugation and subsequent cultivation of chondrocytes (18.6 × 105 cells) in a 96-well plate for adhesion culture led to the production of a scaffold-free cartilage-like cell sheet with a thickness of 0.85 mm. A combined osteochondral-like structure was produced by putting a βTCP block on the cell sheet. The thickness of the cell sheet on the βTCP block and the binding strength between the cell sheet and the βTCP block could be optimized by adjusting the inoculum cell number and timing of βTCP block addition.

Repair of an intercalated long bone defect with a synthetic biodegradable bone-inducing implant

Recombinant human bone morphogenetic protein (rhBMP)-2 in a block copolymer composed of poly- d, l-lactic acid with randomly inserted p-dioxanone and polyethylene glycol (PLA-DX-PEG) as a carrier and porous β-tricalcium phosphate ( β-TCP) blocks were used to generate a new fully absorbable osteogenic biomaterial. The bone regenerability of the rhBMP-2/PLA-DX-PEG/ β-TCP composite was studied in a critical-sized rabbit bone defect model. In an initial study, a composite of PLA-DX-PEG (250 mg) and β-TCP (300 mg) loaded with or without rhBMP2 (50 μg) was implanted into a 1.5 cm intercalated bone defect created in a rabbit femur. Defects were assessed by biweekly radiography until 8 weeks postoperatively. The bony union of the defect was recognized only in the BMP-loaded group. To obtain further data on biomechanical and remodeling properties, another BMP-loaded composites group was made and observed up to 24 weeks. All defects were completely repaired without residual traces of implants. Anatomical and mechanical properties of the repaired bone examined by histology, 3-dimensional CT (3D-CT) and mechanical testing were essentially equivalent to the nonoperated-on femur at 24 weeks. These experimental results indicate that fully absorbable rhBMP-2/PLA-DX-PEG/ β-TCP is a promising composite having osteogenicity efficient enough for repairing large bone defects.

Use of a Biphasic Graft Constructed with Chondrocytes Overlying a β-Tricalcium Phosphate Block in the Treatment of Rabbit Osteochondral Defects

To evaluate the ability of a biphasic construct to repair osteochondral defects in articular cartilage, plugs made of chondrocytes in collagen gel overlying a resorbable porous beta-tricalcium phosphate (TCP) block were implanted into defects in rabbit knees. The repair tissue was evaluated at 8, 12, and 30 weeks. Eight weeks after implantation of the biphasic construct, histologic examination showed hyaline-like cartilage formation that was positive for safranin O and type II collagen. At 12 weeks, most of the beta-TCP was replaced by bone, with a small amount remaining in the underlying cartilage. In the cell-seeded layer, the newly formed middle and deep cartilage adjacent to the subchondral bone stained with safranin O, but no staining was observed in the superficial layer. In addition, cell morphology was distinctly different from the deep levels of the reparative cartilage, with hypertrophic cells at the bottom of the cartilaginous layer. At 30 weeks, beta-TCP had completely resorbed and a tidemark was observed in some areas. In contrast, controls (defects filled with a beta-TCP block alone) showed no cartilage formation but instead had subchondral bone formation. These findings indicate that beta-TCP-supported chondrocytes in collagen gel can partially repair isolated articular cartilage osteochondral defects.

Development of porous apatite ceramic for local delivery of chemotherapeutic agents

An experimental study was conducted on a drug delivery system (DDS), using porous apatite ceramics (PAC): hydroxyapatite block (HAb) [Ca10(PO4)6(OH)2] having a porosity of 35–48% and pore size range of 50–300 μm, and β-tricalcium phosphate block (TCP) [Ca3(PO4)2] having a porosity of 75–80% and pore size range of 100–400 μm, for sustained release of a chemotherapeutic agent. Methotrexate (MTX) was loaded in the pores of PAC blocks by centrifuging the blocks in MTX solution. Impregnation of MTX in PAC blocks (1 cm3) was confirmed by a magnetic resonance imaging (MRI) study using Gadolinium-DTPA enhancement. The MRI showed high signal intensity in the PAC, which was confirmed by dye loading into the pores. To estimate the MTX-releasing capability of the PAC, the blocks were stored in 3 mL of phosphate-buffered saline (PBS) at 37°C and the PBS was replaced every 48 h. The amount of MTX released was assayed by high-performance liquid chromatography. This study showed that MTX-impregnated PAC (0.63–2.25 mg/block) released the drug in a steady manner and maintained its concentration (0.1–1.0 μg/mL) up to 12 days. This concentration is high enough to be effective against tumor cells. Chemotherapeutic agent–impregnated PAC, prepared by simple centrifugation, could be a valuable form of local chemotherapy when used as a strut graft to repair bone defects. This new DDS material could also be used as an adjuvant to extended curettage and provide a means to reduce the recurrence of tumors without risk of systemic toxicity. © 1998 John Wiley & Sons, Inc. J Biomed Mater Res, 39, 536–538, 1998.