Subperiosteal lmplantation of octacalcium phosphate (OCP) stimulates both chondrogenesis and osteogenesis in the tibia,but only osteogenesis in the parietal bone of a rat

Background: It is not known whether long bones and calvaria have distinct biological characteristics. Octacalcium phosphate (OCP), which is a precursor phase of the hydroxyapatite, has been reported to stimulate bone formation if implanted in the subperiosteal region of mouse calvaria. The present study was designed to investigate how the long bone and the calvarium respond to OCP implantation and to compare their biological characteristics. Methods: The synthetic OCP was implanted into the subperiosteal region of rat tibiae and parietal bones being mixed with bovine type I collagen treated by pepsin (Atelocollagen). The biological response was examined histologically and immunohistochemically for collagen matrix phenotypes of types I and II to identify bone and cartilage formation. Results: Both chondrogenesis and osteogenesis were initiated in the tibia 1 week after implantation of OCP and most of the cartilage was replaced by bone at week 2. However, the parietal bone did not show osteogenesis responding to OCP implantation until week 3, and no cartilage formation was associated with the osteogenesis. Conclusions: The present study demonstrated the distinct characteristics of biological response to OCP implantation between the long bone and the calvarium in terms of whether or not cartilage formation is involved in the stimulated osteogenesis by OCP, and in terms of timing of the stimulated chondrogenesis and/or osteogenesis, i.e., the parietal bone takes more time to respond to OCP implantation than the tibia. © 1995 Wiley-Liss, Inc.     

Novel highly biodegradable biphasic tricalcium phosphates composed of alpha-tricalcium phosphate and beta-tricalcium phosphate

Novel biodegradable biphasic tricalcium phosphates (BTCP) composed of alpha-tricalcium phosphate (alpha-TCP) and beta-tricalcium phosphate (beta-TCP) were successfully synthesized by heating amorphous calcium phosphate precursors with different structures at 800 degrees C for 3 h. The ratio of alpha-TCP and beta-TCP in the calcium phosphate particle can be controlled by aging time and pH value during synthesis of the amorphous precursor.     

Effect of self-assembled nanofibrous silk/polycaprolactone layer on the osteoconductivity and mechanical properties of biphasic calcium phosphate scaffolds

We here present the first successful report on combining nanostructured silk and poly(ε-caprolactone) (PCL) with a ceramic scaffold to produce a composite scaffold that is highly porous (porosity ∼85%, pore size ∼500 μm, ∼100% interconnectivity), strong and non-brittle with a surface that resembles extracellular matrix (ECM). The ECM-like surface was developed by self-assembly of nanofibrous structured silk (20-80 nm diameter, similar to native collagen found in ECM) over a thin PCL layer which is coated on biphasic calcium phosphate (BCP) scaffolds. The effects of different concentrations of silk solution on the mechanical and physical properties of the scaffolds were also comprehensively examined. Our results showed that using silk only (irrespective of concentration) for the modification of ceramic scaffolds could drastically reduce the compressive strength of the modified scaffolds in aqueous media, and the modification made a limited contribution to improving scaffold toughness. Using PCL/nanostructured silk the compressive strength and modulus of the modified scaffolds reached 0.42 MPa (compared with 0.07 MPa for BCP) and ∼25 MPa (compared with 5 MPa for BCP), respectively. The failure strain of the modified scaffold increased more than 6% compared with a BCP scaffold (failure strain of less than 1%), indicating a transformation from brittle to elastic behavior. The cytocompatibility of ECM-like composite scaffolds was investigated by studying the attachment, morphology, proliferation and bone-related gene expression of primary human bone-derived cells. Cells cultured on the developed scaffolds for 7 days had significant up-regulation of cell proliferation (∼1.6-fold higher, P < 0.001) and osteogenic gene expression levels (collagen type I, osteocalcin and bone sialoprotein) compared with the other groups tested.     

Calcium phosphate deposition rate,structure and osteoconductivity on electrospun poly(l-lactic acid) matrix using electrodeposition or simulated body fluid incubation

Mineralized nanofibrous scaffolds have been proposed as promising scaffolds for bone regeneration due to their ability to mimic both nanoscale architecture and chemical composition of natural bone extracellular matrix. In this study, a novel electrodeposition method was compared with an extensively explored simulated body fluid (SBF) incubation method in terms of the deposition rate, chemical composition and morphology of calcium phosphate formed on electrospun fibrous thin matrices with a fiber diameter in the range ∼200–1400 nm prepared using 6, 8, 10 and 12 wt.% poly(l-lactic acid) (PLLA) solutions in a mixture of dichloromethane and acetone (2:1 in volume). The effects of the surface modification using the two mineralization techniques on osteoblastic cell (MC3T3-E1) proliferation and differentiation were also examined. It was found that electrodeposition was two to three orders of magnitude faster than the SBF method in mineralizing the fibrous matrices, reducing the mineralization time from ∼2 weeks to 1 h to achieve the same amounts of mineralization. The mineralization rate also varied with the fiber diameter but in opposite directions between the two mineralization methods. As a general trend, the increase of fiber diameter resulted in a faster mineralization rate for the electrodeposition method but a slower mineralization rate for the SBF incubation method. Using the electrodeposition method, one can control the chemical composition and morphology of the calcium phosphate by varying the electric deposition potential and electrolyte temperature to tune the mixture of dicalcium phosphate dihydrate and hydroxyapatite (HAp). Using the SBF method, one can only obtain a low crystallinity HAp. The mineralized electrospun PLLA fibrous matrices from either method similarly facilitate the proliferation and osteogenic differentiation of preosteoblastic MC3T3-E1 cells as compared to neat PLLA matrices. Therefore, the electrodeposition method can be utilized as a fast and versatile technique to fabricate mineralized nanofibrous scaffolds for bone tissue engineering.     

3D printing of composite calcium phosphate and collagen scaffolds for bone regeneration

Low temperature 3D printing of calcium phosphate scaffolds holds great promise for fabricating synthetic bone graft substitutes with enhanced performance over traditional techniques. Many design parameters, such as the binder solution properties, have yet to be optimized to ensure maximal biocompatibility and osteoconductivity with sufficient mechanical properties. This study tailored the phosphoric acid-based binder solution concentration to 8.75 wt% to maximize cytocompatibility and mechanical strength, with a supplementation of Tween 80 to improve printing. To further enhance the formulation, collagen was dissolved into the binder solution to fabricate collagen-calcium phosphate composites. Reducing the viscosity and surface tension through a physiologic heat treatment and Tween 80, respectively, enabled reliable thermal inkjet printing of the collagen solutions. Supplementing the binder solution with 1–2 wt% collagen significantly improved maximum flexural strength and cell viability. To assess the bone healing performance, we implanted 3D printed scaffolds into a critically sized murine femoral defect for 9 weeks. The implants were confirmed to be osteoconductive, with new bone growth incorporating the degrading scaffold materials. In conclusion, this study demonstrates optimization of material parameters for 3D printed calcium phosphate scaffolds and enhancement of material properties by volumetric collagen incorporation via inkjet printing.     

Calcium phosphate cements: action of setting regulators on the properties of the β-tricalcium phosphate-monocalcium phosphate cements

Various additives were tested as setting retarders of the β-tricalcium phosphate-monocalcium phosphate monohydrate (β-TCP-MCPM) cements. Calcium pyrophosphate (CPP), calcium sulphate dihydrate (CSD) and calcium sulphate hemihydrate (CSH) were found to increase the setting time from 30 s to about 10 min. Moreover, the use of CSH resulted in a marked increase of the final diametral strength of the cement, which could be raised from 1MPa to about 3 MPa. The best results were obtained when CSH and CPP were added together to the cement, while the addition of CSD and CPP alone only retarded the setting, without improving the final strength. A particular cement composition (64 wt% β-TCP, 16 wt% MCPM, 15 wt% CSH and 5 wt% CPP), selected for its optimum final strength, was aged in vitro for 8 d at 37°C in saline solution (0.9 wt% NaCl in water). After a moderate decrease, the diametral strength of the specimen was found to level off at about 60% of its initial value (3.2 MPa), for ageing times beyond 1d. This behaviour has been ascribed to the progressive dissolution of the CSD fraction of the hardened cement, compensated by the crystallization of further amounts of DCPD.     

A novel injectable,cohesive and toughened Si-HPMC (silanized-hydroxypropyl methylcellulose) composite calcium phosphate cement for bone substitution

This study reports on the incorporation of the self-setting polysaccharide derivative hydrogel (silanized-hydroxypropyl methylcellulose, Si-HPMC) into the formulation of calcium phosphate cements (CPCs) to develop a novel injectable material for bone substitution. The effects of Si-HPMC on the handling properties (injectability, cohesion and setting time) and mechanical properties (Young’s modulus, fracture toughness, flexural and compressive strength) of CPCs were systematically studied. It was found that Si-HPMC could endow composite CPC pastes with an appealing rheological behavior at the early stage of setting, promoting its application in open bone cavities. Moreover, Si-HPMC gave the composite CPC good injectability and cohesion, and reduced the setting time. Si-HPMC increased the porosity of CPCs after hardening, especially the macroporosity as a result of entrapped air bubbles; however, it improved, rather than compromised, the mechanical properties of composite CPCs, which demonstrates a strong toughening and strengthening effect. In view of the above, the Si-HPMC composite CPC may be particularly promising as bone substitute material for clinic application.     

β-TCP植入人体骨缺损后的组织学观察

目的了解多孔β—TCP在植入人体后的的成骨变化及降解过程。方法对12例多孔β—TCP活检标本行不脱钙硬组织切片检查观察陶瓷周围和内部的新生组织、陶瓷的形态改变、降解颗粒及伴随的细胞吞噬反应。其中11例标本行组织形态计量。结果所有的β—TCP标本均可见新生骨组织，新骨与材料紧密接触；材料的结构变得稀疏，周围可见降解颗粒和吞噬细胞。新生骨组织（VPB）为21．83％，材料残余率为21．17％。结论多孔β—TCP是一种良好的骨传导材料，植入体内后能逐步降解并形成新生骨组织。陶瓷产生降解颗粒及诱发的细胞吞噬反应应引起注意。     

Umbilical cord and bone marrow mesenchymal stem cell seeding on macroporous calcium phosphate for bone regeneration in rat cranial defects

Human umbilical cord mesenchymal stem cells (hUCMSCs) are inexhaustible and can be harvested at a low cost without an invasive procedure. However, there has been no report on comparing hUCMSCs with human bone marrow MSCs (hBMSCs) for bone regeneration in vivo. The aim of this study was to investigate hUCMSC and hBMSC seeding on macroporous calcium phosphate cement (CPC), and to compare their bone regeneration in critical-sized cranial defects in rats. Cell attachment, osteogenic differentiation and mineral synthesis on RGD-modified macroporous CPC were investigated in vitro. Scaffolds with cells were implanted in 8-mm defects of athymic rats. Bone regeneration was investigated via micro-CT and histological analysis at 4, 12, and 24 weeks. Three groups were tested: CPC with hUCMSCs, CPC with hBMSCs, and CPC control without cells. Percentage of live cells and cell density on CPC in vitro were similarly good for hUCMSCs and hBMSCs. Both cells had high osteogenic expressions of alkaline phosphatase, osteocalcin, collagen I, and Runx2. Bone mineral density and trabecular thickness in hUCMSC and hBMSC groups in vivo were greater than those of CPC control group. New bone amount for hUCMSC-CPC and hBMSC-CPC constructs was increased by 57% and 88%, respectively, while blood vessel density was increased by 15% and 20%, than CPC control group at 24 weeks. hUCMSC-CPC and hBMSC-CPC groups generally had statistically similar bone mineral density, new bone amount and vessel density. In conclusion, hUCMSCs seeded on CPC were shown to match the bone regeneration efficacy of hBMSCs in vivo for the first time. Both hUCMSC-CPC and hBMSC-CPC constructs generated much more new bone and blood vessels than CPC without cells. Macroporous RGD-grafted CPC with stem cell seeding is promising for craniofacial and orthopedic repairs.     

Effect of some physico-chemical properties of matrix on lengthwise and oriented growth of octacalcium phosphate crystal

Relationship between some physicochemical properties of matrix, such as viscosity, density or structure of the framework, and crystal growth of octacalcium phosphate (OCP) was studied using various concentrations of polyacrylamide gels at 37 degrees C and at pH 6.5. Reaction was carried out in a model system of enamel formation, where calcium solution (30 mM) and phosphate solution (5 mM) were separated by a cation selective membrane with polyacrylamide gel on the PO4 side. OCP grew in ribbon-like morphology in 5-20% polyacrylamide gels. Crystal size of OCP decreased with an increase in gel concentration. In 30% gel, growth of OCP was disturbed, In 7.5% polyacrylamide gel, 1% albumin reduced the crystal size of OCP. In contrast, 1% enamel proteins did not much reduce the crystal size of OCP and OCP crystallized in its characteristic ribbon-like morphology.     

Repair of rabbit segmental femoral defects by using a combination of tetrapod-shaped calcium phosphate granules and basic fibroblast growth factor-binding ion complex gel

The effect of tetrapod-shaped alpha tricalcium phosphate granules (TB) as a scaffold combined with basic fibroblast growth factor (bFGF)-binding ion complex gel (f-IC gel) on neovascularization and bone regeneration was evaluated in segmental femoral defects of rabbits. The defects were stabilized using a plate with a polypropylene mesh cage (PMC) containing one of the following: PMC alone (PMC group), TB (TB group), TB and bFGF (TB/f group), TB and IC gel (TB/IC group), or TB and f-IC gel (TB/f-IC group). Four rabbits from each group were euthanized at 2 and 4 weeks after surgery. Histomorphometry showed that the number of vessels and the volume of new bone in the TB/f-IC group were significantly higher than those in the other groups at all time points. There were no differences in the extent of neovascularization and new bone formation between the TB and TB/f groups. These findings suggest that the combination of TB and f-IC gel facilitated both neovascularization and new bone formation in segmental femoral defects of rabbits. This combination may be of considerable use for treating segmental long bone defects.     

Human embryonic stem cells and macroporous calcium phosphate construct for bone regeneration in cranial defects in rats

Human embryonic stem cells (hESCs) are an exciting cell source as they offer an unlimited supply of cells that can differentiate into all cell types for regenerative medicine applications. To date, there has been no report on hESCs with calcium phosphate cement (CPC) scaffolds for bone regeneration in vivo. The objectives of this study were to: (i) investigate hESCs for bone regeneration in vivo in critical-sized cranial defects in rats; and (ii) determine the effects of cell seeding and platelets in macroporous CPC on new bone and blood vessel formation. hESCs were cultured to yield mesenchymal stem cells (MSCs), which underwent osteogenic differentiation. Four groups were tested in rats: (i) CPC control without cells; (ii) CPC with hESC-derived MSCs (CPC + hESC-MSC); (iii) CPC with hESC-MSCs and 30% human platelet concentrate (hPC) (CPC + hESC-MSC + 30% hPC); and (iv) CPC + hESC-MSC + 50% hPC. In vitro, MSCs were derived from embryoid bodies of hESCs. Cells on CPC were differentiated into the osteogenic lineage, with highly elevated alkaline phosphatase and osteocalcin expressions, as well as mineralization. At 12 weeks in vivo, the groups with hESC-MSCs and hPC had three times as much new bone as, and twice the blood vessel density of, the CPC control. The new bone in the defects contained osteocytes and blood vessels, and the new bone front was lined with osteoblasts. The group with 30% hPC and hESC-MSCs had a blood vessel density that was 49% greater than the hESC-MSC group without hPC, likely due to the various growth factors in the platelets enhancing both new bone and blood vessel formation. In conclusion, hESCs are promising for bone tissue engineering, and hPC can enhance new bone and blood vessel formation. Macroporous CPC with hESC-MSCs and hPC may be useful for bone regeneration in craniofacial and orthopedic applications.     

Synergistic effects of bisphosphonate and calcium phosphate nanoparticles on peri-implant bone responses in osteoporotic rats

The prevalence of osteoporosis will increase within the next decades due to the aging world population, which can affect the bone healing response to dental and orthopedic implants. Consequently, local drug targeting of peri-implant bone has been proposed as a strategy for the enhancement of bone-implant integration in osteoporotic conditions. In the present study, an established in-vivo femoral condyle implantation model in osteoporotic and healthy bone is used to analyze the osteogenic capacity of titanium implants coated with bisphosphonate (BP)-loaded calcium phosphate nanoparticles (nCaP) under compromised medical conditions. After 4 weeks of implantation, peri-implant bone volume (%BV; by μCT) and bone area (%BA; by histomorphometry) were significantly increased within a distance of 500 μm from implant surfaces functionalized with BP compared to control implants in osteoporotic and healthy conditions. Interestingly, the deposition of nCaP/BP coatings onto implant surfaces increased both peri-implant bone contact (%BIC) and volume (%BV) compared to the deposition of nCaP or BP coatings individually, in osteoporotic and healthy conditions. The results of real-time PCR revealed similar osteogenic gene expression levels to all implant surfaces at 4-weeks post-implantation. In conclusion, simultaneous targeting of bone formation (by nCaP) and bone resorption (by BP) using nCaP/BP surface coatings represents an effective strategy for synergistically improvement of bone-implant integration, especially in osteoporotic conditions.     

多孔磷酸三钙修复良性骨肿瘤骨缺损的临床研究

目的评价多孔磷酸三钙(Tricalcium Phosphate,TCP)治疗良性或侵袭性骨肿瘤骨缺损临床效果。方法 2007年9月至2010年1月对19例良性或侵袭性骨肿瘤患者采用多孔磷酸三钙修复骨缺损,其中男12例,女7例。年龄3~58岁,平均年龄22.4岁。随访3~30月,平均13月。采用VAS评分反应疼痛缓解程度,X线检查并Lane-Sandhu评分评定骨愈合情况,分析X片中植骨区灰度变化评估TCP的降解情况。结果 VAS评分术前、术后1周、术后12周、术后24周各组间总体差别比较<0.01,说明术后疼痛逐渐缓解。Lane-sandhu评分三个时段差异<0.001,说明人工骨随时间推移有明显的骨修复效果。认为手术中应当将多孔TCP人工骨松散放置,不要施加任何挤压力。结论多孔磷酸三钙人工骨可以用作良性或侵袭性骨肿瘤骨缺损的修复。     

Synthesis of a novel b-tricalcium phosphate/hydroxyapatite biphasic calcium phosphate containing niobium ions and evaluation of its osteogenic properties

To promote the osteogenic properties of osteoblasts, we synthesized a hydroxyapatite (HAp) with β-tricalcium phosphate (β-TCP) biphasic calcium phosphate containing Nb ions (NbTCP/HAp). NbTCP/HAp was prepared by annealing precipitates obtained by coprecipitation of an aqueous solution of Ca(NO₃)₂ and a mixture of (NH₄)₂HPO₄ and aqueous Nb solution. The precipitates can be regarded as a calcium-deficient HAp, the PO₄ sites of which are partly occupied by Nb ions. NbTCP/HAp was successfully synthesized by thermal decomposition of the precipitates. NbTCP/HAp enhanced the calcification of normal human osteoblasts (NHOst), and the amount of calcified tissue increased in proportion to the Nb ion concentration in the NbTCP/HAp. The alkaline phosphatase (ALP) activity of NHOst was also enhanced by NbTCP/HAp. Because Nb ions significantly enhance the ALP activity of NHOst, calcification by NbTCP/HAp is considered to be due to enhancement of ALP activity induced by Nb ions dissolved from NbTCP/HAp. These results indicate that NbTCP/HAp can be an effective bone repair material.     

Self-healing hybrid nanocomposites consisting of bisphosphonated hyaluronan and calcium phosphate nanoparticles

Non-covalent interactions are often regarded as insufficient to construct macroscopic materials of substantial integrity and cohesion. However, the low binding energy of such reversible interactions can be compensated by increasing their number to work in concert to create strong materials. Here we present the successful development of an injectable, cohesive nanocomposite hydrogel based on reversible bonds between calcium phosphate nanoparticles and bisphosphonate-functionalized hyaluronic acid. These nanocomposites display a capacity for self-healing as well as adhesiveness to mineral surfaces such as enamel and hydroxyapatite. Most importantly, these non-covalently cross-linked composites are surprisingly robust yet biodegradable upon extensive in vitro and in vivo testing and show bone interactive capacity evidenced by bone ingrowth into material remnants. The herein presented method provides a new methodology for constructing nanoscale composites for biomedical applications, which owe their integrity to reversible bonds.     

Effect of polyethylene pretreatments on the biomimetic deposition and adhesion of calcium phosphate films

The effect of ultraviolet irradiation and glow discharge (GD) processing of the polyethylene (PE) substrates on deposition of calcium phosphate (CaP) films from supersaturated aqueous calcium phosphate solutions was investigated in this study. CaP coatings deposited on the PE substrates were comprised of elongated clusters of spherical particles and 100% of the free surface area of nearly all of the substrates was covered with a porous CaP film after a 3 day immersion. Nano-scratch tests determined that PE-CaP adhesion was most improved when PE substrates were subjected to 50W GD treatments. As determined by contact angle measurements, the GD-treated PE samples had the highest electron donor parameter of surface energy, suggesting that enhancing the electron donor parameter of PE leads to improved adhesion with the biomimetic CaP coating.     

Effect of phosphate functional groups on the calcification capacity of acrylic hydrogels

The incorporation of negatively charged groups into the structure of synthetic polymers is frequently advocated as a method for enhancing their calcification capacity required in orthopedic and dental applications. However, the results reported by various research groups are rather contentious, since inhibitory effects have also been observed in some studies. In the present study, phosphate groups were introduced in poly(2-hydroxyethyl methacrylate) (PHEMA) by copolymerization with 10% mol of either mono(2-acryloyloxyethyl) phosphate (MAEP) or mono(2-methacryloyloxyethyl) phosphate (MMEP). Incubation of these hydrogels for determined durations (1-9 weeks) in a simulated body fluid (SBF) solution induced deposition of calcium phosphate (CaP) deposits of whitlockite type. After 9 weeks, the amount of calcium deposited on the phosphate-containing polymers was four times lower than that found on PHEMA, as determined by X-ray photoelectron spectroscopy (XPS). Samples of copolymer HEMA-MAEP were implanted subcutaneously in rats and evaluated after 9 weeks. No CaP deposits could be detected on the copolymer by XPS or energy dispersive X-ray spectroscopy, while PHEMA samples were massively calcified. It was concluded that the presence of phosphate groups decreased the calcification capacity of the hydrogels, and that in the conditions of this study, the phosphate groups had an inhibitory effect on the deposition of CaP phases on HEMA-based hydrogels.