Injectable PLGA microsphere/calcium phosphate cements: physical properties and degradation characteristics

Calcium phosphate (CaP) cements show an excellent biocompatibility and often have a high mechanical strength, but in general degrade relatively slow. To increase degradation rates, macropores can be introduced into the cement, e.g., by the inclusion of biodegradable microspheres into the cement. The aim of this research is to develop an injectable PLGA microsphere/CaP cement with sufficient setting/cohesive properties and good mechanical and physical properties. PLGA microspheres were prepared using a water-in-oil-in-water double-emulsion technique. The CaP-cement used was Calcibon, a commercially available hydroxyapatite-based cement. 10:90 and 20:80 dry wt% PLGA microsphere/CaP cylindrical scaffolds were prepared as well as microporous cement (reference material). Injectability, setting time, cohesive properties and porosity were determined. Also, a 12-week degradation study in PBS (37 degree C) was performed. Results showed that injectability decreased with an increase in PLGA microsphere content. Initial and final setting time of the PLGA/CaP samples was higher than the microporous sample. Porosity of the different formulations was 40.8% (microporous), 60.2% (10:90) and 69.3% (20:80). The degradation study showed distinct mass loss and a pH decrease of the surrounding medium starting from week 6 with the 10:90 and 20:80 formulations, indicating PLGA erosion. Compression strength of the PLGA microsphere/CaP samples decreased siginificantly in time, the microporous sample remained constant. After 12 weeks both PLGA/CaP samples showed a structure of spherical micropores and had a compressive strength of 12.2 MPa (10:90) and 4.3 MPa (20:80). Signs of cement degradation were also found with the 20:80 formulation. In conclusion, all physical parameters were well within workable ranges with both 10:90 and 20:80 PLGA microsphere/CaP cements. After 12 weeks the PLGA was totally degraded and a highly porous, but strong scaffold remained.     

The effect of porosity on drug release kinetics from vancomycin microsphere/calcium phosphate cement composites

The influence of porosity on release profiles of antibiotics from calcium phosphate composites was investigated to optimize the duration of treatment. We hypothesized, that by the encapsulation of vancomycin-HCl into biodegradable microspheres prior admixing to calcium phosphate bone cement, the influence of porosity of the cement matrix on vancomycin release could be reduced. Encapsulation of vancomycin into a biodegradable poly(lactic co-glycolic acid) copolymer (PLGA) was performed by spray drying; drug-loaded microparticles were added to calcium phosphate cement (CPC) at different powder to liquid ratios (P/L), resulting in different porosities of the cement composites. The effect of differences in P/L ratio on drug release kinetics was compared for both the direct addition of vancomycin-HCl to the cement liquid and for cement composites modified with vancomycin-HCl-loaded microspheres. Scanning electron microscopy (SEM) was used to visualize surface and cross section morphology of the different composites. Brunauer, Emmett, and Teller-plots (BET) was used to determine the specific surface area and pore size distribution of these matrices. It could be clearly shown, that variations in P/L ratio influenced both the porosity of cement and vancomycin release profiles. Antibiotic activity during release study was successfully measured using an agar diffusion assay. However, vancomycin-HCl encapsulation into PLGA polymer microspheres decreased porosity influence of cement on drug release while maintaining antibiotic activity of the embedded substance.     

In vitro degradation rate of apatitic calcium phosphate cement with incorporated PLGA microspheres

Calcium phosphate cements (CPCs) are frequently used as bone substitute material. Despite their superior clinical handling and excellent biocompatibility, they exhibit poor degradability, which limits bone ingrowth into the implant. Microspheres were prepared from poly(d,l-lactic-co-glycolic acid) (PLGA) and included in injectable CPCs as porogens in order to enhance its macroporosity after the polymeric microspheres had degraded. Upon degradation of the PLGA microspheres, acid is produced that enhances the dissolution rate of the CPC. However, the effect of the characteristics of PLGA microspheres on the degradation rate of CPCs has never been studied before. Therefore, the purpose of the current study was to investigate the dependence of CPC degradation on the chemical and morphological characteristics of incorporated PLGA microspheres. With respect to the chemical characteristics of the PLGA microspheres, the effects of both PLGA molecular weight (5, 17 and 44kDa) and end-group functionalization (acid-terminated or end-capped) were studied. In addition, two types of PLGA microspheres, differing in morphology (hollow vs. dense), were tested. The results revealed that, although both chemical parameters clearly affected the polymer degradation rate when embedded as hollow microspheres in CPC, the PLGA and CPC degradation rates were mainly dependent on the end-group functionalization. Moreover, it was concluded that dense microspheres were more efficient porogens than hollow ones by increasing the CPC macroporosity during in vitro incubation. By combining all test parameters, it was concluded that dense PLGA microspheres consisting of acid-terminated PLGA of 17kDa exhibited the highest and fastest acid-producing capacity and correspondingly the highest and fastest amount of porosity. In conclusion, the data presented here indicate that the combination of dense, acid-terminated PLGA microspheres with CPC emerges as a successful combination to achieve enhanced apatitic CPC degradation.     

Evaluation of the biocompatibility of calcium phosphate cement/PLGA microparticle composites

In this study, the biocompatibility of a calcium phosphate (CaP) cement incorporating poly (D,L-lactic-co-glycolic acid) (PLGA) microparticles was evaluated in a subcutaneous implantation model in rats. Short-term biocompatibility was assessed using pure CaP discs and CaP discs incorporating PLGA microparticles (20% w/w) with and without preincubation in water. Long-term biocompatibility was assessed using CaP discs incorporating varying amounts (5, 10, or 20% w/w) and diameter sizes (small, 0-50 mum; medium, 51-100 mum, or large, 101-200 mum) of PLGA microparticles. The short-term biocompatibility results showed a mild tissue response for all implant formulations, irrespective of disc preincubation, during the early implantation periods up to 12 days. Quantitative histological evaluation revealed that the different implant formulations induced the formation of similar fibrous tissue capsules and interfaces. The results concerning long-term biocompatibility showed that all implants were surrounded by a thin connective tissue capsule (<10 layers of fibroblasts). Additionally, no significant differences in capsule and interface scores were observed between the different implant formulations. The implants containing 20% PLGA with medium- and large-sized microparticles showed fibrous tissue ingrowth throughout the implants, indicating PLGA degradation and interconnectivity of the pores. The results demonstrate that CaP/PLGA composites evoke a minimal inflammatory response. The implants containing 20% PLGA with medium- and large-sized microparticles showed fibrous tissue ingrowth after 12- and 24-weeks indicating PLGA degradation and interconnectivity of the pores. Therefore, CaP/PLGA composites can be regarded as biocompatible biomaterials with potential for bone tissue engineering and advantageous possibilities of the microparticles regarding material porosity.     

Preparation of macroporous calcium phosphate cement tissue engineering scaffold

Unlike sintered hydroxyapatite there is evidence to suggest that calcium phosphate cement (CPC) is actively remodelled in vivo and because CPC is formed by a low-temperature process, thermally unstable compounds such as proteins may be incorporated into the matrix of the cement which can then be released after implantation. The efficacy of a macroporous CPC as a bone tissue engineering scaffold has been reported; however, there have been few previous studies on the effect of macroporosity on the mechanical properties of the CPC. This study reports a novel method for the formation of macroporous CPC scaffolds, which has two main advantages over the previously reported manufacturing route: the cement matrix is considerably denser than CPC formed from slurry systems and the scaffold is formed at temperatures below room temperature. A mixture of frozen sodium phosphate solution particles and CPC powder were compacted at 106 MPa and the sodium phosphate was allowed to melt and simultaneously set the cement. The effect of the amount of porogen used during processing on the porosity, pore size distribution and compressive strength of the scaffold was investigated. It was found that macroporous CPC could reliably be fabricated using cement:ice ratios as low as 5:2.     

载药型磷酸钙骨水泥的制备及体外释放性能

背景：磷酸钙骨水泥具有良好的生物相容性,可作为骨修复材料与药物载体。 目的：制备载药磷酸三钙骨水泥,并分析其体外释放性能。 方法：采用共沉淀法制备磷酸三钙前躯体,经高温煅烧研磨获得α-磷酸三钙粉体,测试含不同浓度(1.25%,2.5%,3.75%,5%)抗生素(头孢拉定或头孢氨苄或环丙沙星)骨水泥,浸泡不同时间后(6 h、12 h、24 h、2 d、3 d、4 d、 5 d、6 d、7 d、8 d)的药物体外释放浓度。 结果与结论：制备的磷酸三钙粉体粒度约2 μm,结晶度良好。载不同抗生素的骨水泥体外释放都受自身物理性质的影响。载药骨水泥中环丙沙星能够满足长时间缓释,并能达到一个比较理想的缓释浓度,头孢类药物由于自身稳定性等原因,缓释效果并不理想。头孢氨苄的水解速率较低,环丙沙星的光降解条件比较苛刻,因此两者释放未受太大影响,与Higuchi模型基本吻合;头孢拉定的水解速率相对较高,对体系的释放驱动力产生较大影响,使得释放不再遵循Higuchi模型。     

Micro-architecture of calcium phosphate granules and fibrin glue composites for bone tissue engineering

Calcium phosphate ceramics are currently used as bone graft substitutes in various types of clinical applications. Fibrin glue is also used in surgery due to its haemostatic, chemotactic and mitogenic properties. By combining these two biomaterials, new composite scaffolds were prepared. In this study, we attempt to analyse whether thrombin concentration in the fibrin glue could influence the properties of the composite. The association between fibrin glue and calcium phosphate ceramic granules was characterized at the ultra structural level. Micro and macroporous biphasic calcium phosphate ceramic granules with a diameter of 1-2mm composed of hydroxyapatite and beta-tricalcium phosphate (60/40) were associated to fibrin glue. The composites were observed by scanning and transmission electron microscopy and microcomputed tomography. Fibre thickness, porosity and homogeneity of the fibrin clot were modified by increased the thrombin concentration. Mixing fibrin glue with calcium phosphate granules (1:2) did not modify the microstructure of the fibrin clot in the composite. Nevertheless, thrombin interacted with the bioceramic by inducing the nucleation of crystalline precipitate at the ceramic/fibrin glue interface. Combining fibrin sealant and calcium phosphate ceramics could lead to new scaffolds for bone tissue engineering with the synergy of the properties of the two biomaterials.     

Biocompatibility and degradation of poly(DL-lactic-co-glycolic acid)/calcium phosphate cement composites

Injectable calcium phosphate (Ca-P) cement materials exhibit favorable osteocompatible behavior but are resorbed slowly because of a lack of a bone ingrowth-enabling macroporosity. In this study, poly(DL-lactic-co-glycolic acid) (PLGA) microparticles (average size 66 +/- 25 microm) were incorporated into Ca-P cement to obtain a macroporous Ca-P cement scaffold after PLGA hydrolysis in vivo. Preset PLGA/Ca-P cement composite discs of various weight ratios (0/100, 15/85, 30/70, and 50/50) were implanted subcutaneously and in cranial defects in rats for 12 weeks. Histological analysis revealed that all macropores in the PLGA-containing composites (average pore size 73 +/- 27 microm) were filled with fibrous tissue and blood vessels (subcutaneous implants) and/or bone (cranial implants). Histologically, bone formation appeared most abundant and most consistent in the 30/70 PLGA/Ca-P cement composites. Histomorphometrical evaluation revealed a significant increase in defect fill in the 15/85 and 30/70 PLGA/Ca-P cement composites. Finally, subcutaneous and cranial 50/50 PLGA/Ca-P cement composites had degraded to a large extent, without adequate replacement by bone in the cranial implants. Therefore, we conclude that PLGA/Ca-P cement composites enable tissue ingrowth and show excellent osteocompatibility in weight ratios of 15/85 and 30/70 PLGA/Ca-P cement. In this model, 30/70 PLGA/Ca-P cement composites showed the most favorable biological response.     

In vitro evaluation of potential calcium phosphate scaffolds for tissue engineering

Nanocrystalline calcium phosphates are very interesting candidates as scaffolds for bone tissue engineering. These materials show excellent in vivo biocompatibility, cell proliferation, and resorption. In this work we have studied the osteoblast-like cell behavior seeded onto HA and BCP synthesized by controlled crystallization method and treated at different temperatures. In vitro cell attachment, proliferation, differentiation, spreading, and cytotoxicity tests have been carried out. The results can be explained as a function of the phase composition and microstructure. Under in vitro closed conditions, nanocrystalline HA depletes the calcium of the medium avoiding cell proliferation, whereas well-crystallized HA enhances high cell proliferation. On the other hand, nanocrystalline BCPs supply Ca(2+) to the medium due to the higher solubility of the beta-TCP component, allowing an excellent in vitro cellular response when osteoblast-like cells are seeded on it. These features make BCPs excellent candidates as scaffolds for bone tissue engineering.     

In vitro characterization of hepatocyte growth factor release from PHBV/PLGA microsphere scaffold

Polymer scaffolds which can support cells to grow as well as deliver growth factors to the cells simultaneously have great potential for the successful regeneration of failed tissues. As popularly used vehicles to deliver anti-cancer drugs and growth factors, microspheres also show many advantages as substrates to guide the growth of cells. Therefore, we aimed to examine the feasibility of using microspheres as ideal scaffolds for liver tissue engineering. To determine the capabilities of previously used microsphere scaffold to deliver growth factors simultaneously, this work investigated a long-term (about three months) release of bovine serum albumin (BSA) from microsphere scaffolds fabricated by using two different polymers, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV, 8% PHV), poly(lactide-co-glycolide) acid (PLGA, 5050) and a blend of PLGA and PHBV. BSA served as a model for hepatocyte growth factor (HGF) since both proteins have similar molecular weights and hydrophilicity. Furthermore, HGF was encapsulated into the PLGA/PHBV composite microsphere with a core-shell structure, and sustained delivery of HGF with maintained bioactivity was achieved for at least 40 days. The moderate degradation rate (about 55% loss of the initial mass) and well-preserved structure after three months of incubation indicated that the PLGA/PHBV composite microspheres would therefore be more suitable than the pure PHBV or PLGA microspheres as a scaffold for engineering liver tissue.     

Poly(propylene fumarate) reinforced dicalcium phosphate dihydrate cement composites for bone tissue engineering

Calcium phosphate cements have many desirable properties for bone tissue engineering, including osteoconductivity, resorbability, and amenability to rapid prototyping-based methods for scaffold fabrication. In this study, we show that dicalcium phosphate dihydrate (DCPD) cements, which are highly resorbable but also inherently weak and brittle, can be reinforced with poly(propylene fumarate) (PPF) to produce strong composites with mechanical properties suitable for bone tissue engineering. Characterization of DCPD-PPF composites revealed significant improvements in mechanical properties for cements with a 1.0 powder to liquid ratio. Compared with nonreinforced controls, flexural strength improved from 1.80 ± 0.19 MPa to 16.14 ± 1.70 MPa, flexural modulus increased from 1073.01 ± 158.40 MPa to 1303.91 ± 110.41 MPa, maximum displacement during testing increased from 0.11 ± 0.04 mm to 0.51 ± 0.09 mm, and work of fracture improved from 2.74 ± 0.78 J/m(2) to 249.21 ± 81.64 J/m(2) . To demonstrate the utility of our approach for scaffold fabrication, 3D macroporous scaffolds were prepared with rapid prototyping technology. Compressive testing revealed that PPF reinforcement increased scaffold strength from 0.31 ± 0.06 MPa to 7.48 ± 0.77 MPa. Finally, 3D PPF-DCPD scaffolds were implanted into calvarial defects in rabbits for 6 weeks. Although the addition of mesenchymal stem cells to the scaffolds did not significantly improve the extent of regeneration, numerous bone nodules with active osteoblasts were observed within the scaffold pores, especially in the peripheral regions. Overall, the results of this study suggest that PPF-DCPD composites may be promising scaffold materials for bone tissue engineering.     

Biocompatibility and osteogenicity of degradable Ca-deficient hydroxyapatite scaffolds from calcium phosphate cement for bone tissue engineering

Ca-deficient hydroxyapatite (CDHA) porous scaffolds were successfully fabricated from calcium phosphate cement (CPC) by a particle-leaching method. The morphology, porosity and mechanical strength as well as degradation of the scaffolds were characterized. The results showed that the CDHA scaffolds with a porosity of 81% showed open macropores with pore sizes of 400-500mum. Thirty-six per cent of these CDHA scaffolds were degraded after 12 weeks in Tris-HCl solution. Mesenchymal stem cells (MSCs) were cultured, expanded and seeded on the scaffolds, and the proliferation and differentiation of MSCs into osteoblastic phenotype were determined using MTT assay, alkaline phosphatase activity and scanning electron microscopy. The results revealed that the CDHA scaffolds were biocompatible and had no negative effects on the MSCs in vitro. The in vivo biocompatibility and osteogenicity of the scaffolds were investigated. Both CDHA scaffolds and MSC/scaffold constructs were implanted in rabbit mandibles and studied histologically. The results showed that CDHA scaffolds exhibited good biocompatibility and osteoconductivity. Moreover, the introduction of MSCs into the scaffolds dramatically enhanced the efficiency of new bone formation, especially at the initial stage after implantation (from 2 to 4 weeks). However, the CDHA scaffolds showed as good biocompatibility and osteogenicity as the hybrid ones at 8 weeks. These results indicate that the CDHA scaffolds fulfill the basic requirements of bone tissue engineering scaffold.     

The sintered microsphere matrix for bone tissue engineering: in vitro osteoconductivity studies

A tissue engineering approach has been used to design three-dimensional synthetic matrices for bone repair. The osteoconductivity and degradation profile of a novel polymeric bone-graft substitute was evaluated in an in vitro setting. Using the copolymer poly(lactide-co-glycolide) [PLAGA], a sintering technique based on microsphere technology was used to fabricate three-dimensional porous scaffolds for bone regeneration. Osteoblasts and fibroblasts were seeded onto a 50:50 PLAGA scaffold. Morphologic evaluation through scanning electron microscopy demonstrated that both cell types attached and spread over the scaffold. Cells migrated through the matrix using cytoplasmic extensions to bridge the structure. Cross-sectional images indicated that cellular proliferation had penetrated into the matrix approximately 700 microm from the surface. Examination of the surfaces of cell/matrix constructs demonstrated that cellular proliferation had encompassed the pores of the matrix by 14 days of cell culture. With the aim of optimizing polymer composition and polymer molecular weight, a degradation study was conducted utilizing the matrix. The results demonstrate that degradation of the sintered matrix is dependent on molecular weight, copolymer ratio, and pore volume. From this data, it was determined that 75:25 PLAGA with an initial molecular weight of 100,000 has an optimal degradation profile. These studies show that the sintered microsphere matrix has an osteoconductive structure capable of functioning as a cellular scaffold with a degradation profile suitable for bone regeneration.     

Poly(propylene fumarate)/(calcium sulphate/beta-tricalcium phosphate) composites: preparation, characterization and in vitro degradation

This study aimed to prepare a poly(propylene fumarate)/(calcium sulphate/beta-tricalcium phosphate) (PPF/(CaSO(4)/beta-TCP)) composite. We first examined the effects of varying the molecular weight of PPF and the N-vinyl pyrrolidinone (NVP) to PPF ratio on the maximum cross-linking temperature and the composite compressive strength and modulus. Then the in vitro biodegradation behaviour of PPF/(CaSO(4)/beta-TCP) composites was investigated. The effects of varying the molecular weight of PPF, the NVP/PPF ratio and the CaSO(4)/beta-TCP molar ratio on the weight loss and the composite compressive strength and modulus were examined. The cross-linking temperature, which increased with increasing molecular weight of PPF and NVP/PPF ratio, ranged from 41 to 43 degrees C for all formulations. The mechanical properties were increased by a decrease in the NVP/PPF ratio. For all formulations, the compressive strength values fell between 12 and 62 MPa, while the compressive modulus values fell between 290 and 1149 MPa. The weight loss decreased either with increasing molecular weight of PPF or with decreasing NVP/PPF ratio and CaSO(4)/beta-TCP molar ratio during degradation. The compressive strength and modulus increased with decreasing NVP/PPF ratio or decreasing CaSO(4)/beta-TCP ratio. The greatest weight loss over 6 weeks was 14.72%. For all formulations, the compressive modulus values fell between 57 and 712 MPa and the compressive strength fell between 0.5 and 21 MPa throughout 6 weeks degradation. Scanning electron microscopy and X-ray diffraction analysis of the PPF/(CaSO(4)/beta-TCP) composites demonstrated that hydroxyapatite was deposited on the surface of CaSO(4)/beta-TCP granules during degradation.     

Human embryonic stem cell encapsulation in alginate microbeads in macroporous calcium phosphate cement for bone tissue engineering

Human embryonic stem cells (hESC) are promising for use in regenerative medicine applications because of their strong proliferative ability and multilineage differentiation capability. To date there have been no reports on hESC seeding with calcium phosphate cement (CPC). The objective of this study was to investigate hESC-derived mesenchymal stem cell (hESCd-MSC) encapsulation in hydrogel microbeads in macroporous CPC for bone tissue engineering. hESC were cultured to form embryoid bodies (EB), and the MSC were then migrated out of the EB. hESCd-MSC had surface markers characteristic of MSC, with positive alkaline phosphatase (ALP) staining when cultured in osteogenic medium. hESCd-MSC were encapsulated in alginate at a density of 1millioncellsml(-1), with an average microbead size of 207μm. CPC contained mannitol porogen to create a porosity of 64% and 218-μm macropores, with 20% absorbable fibers for additional porosity when the fibers degrade. hESCd-MSC encapsulated in microbeads in CPC had good viability from 1 to 21days. ALP gene expression at 21days was 25-fold that at 1day. Osteocalcin (OC) at 21days was two orders of magnitude of that at 1day. ALP activity in colorimetric p-nitrophenyl phosphate assay at 21days was fivefold that at 1day. Mineral synthesis by the encapsulated hESCd-MSC at 21days was sevenfold that at 1day. Potential benefits of the CPC-stem cell paste include injectability, intimate adaptation to complex-shaped bone defects, ease in contouring to achieve esthetics in maxillofacial repairs, and in situ setting ability. In conclusion, hESCd-MSC were encapsulated in alginate microbeads in macroporous CPC, showing good cell viability, osteogenic differentiation and mineral synthesis for the first time. The hESCd-MSC-encapsulating macroporous CPC construct is promising for bone regeneration in a wide range of orthopedic and maxillofacial applications.     

Alginate/poly (lactic-co-glycolic acid)/calcium phosphate cement scaffold with oriented pore structure for bone tissue engineering

In this study, the alginate/calcium phosphate cement (CPC) scaffolds with oriented pore structure were fabricated by unidirectional freeze casting and poly (lactic-co-glycolic acid) (PLGA) was used to infiltrate into the macropores to strengthen the scaffolds. By modifying the liquid to powder ratio, the porosity and pore size of the alginate/CPC scaffold could be controlled. At the liquid to powder (L/P) ratio of 3.25, scaffolds possessing open directional macropores and a total porosity of 89.24% could be achieved. The size of the tubule-like macropores could reach 100-200 mum in their radial dimension and more than 1000 mum in the axial one, with macropores well-regulated arrayed. Increasing the L/P ratio would significantly decrease the mechanical strength of alginate/CPC scaffolds. The compressive strength and toughness of scaffolds could be greatly improved via PLGA reinforcement. Three mechanisms of PLGA reinforcement ran as follows: participating in the external load, strengthening the matrix, and patching the defects of CPC pores wall. Alginate/PLGA/CPC scaffold preserved the open directional macropores and might be a potential scaffold for bone tissue engineering.     

Direct deposited porous scaffolds of calcium phosphate cement with alginate for drug delivery and bone tissue engineering

This study reports the preparation of novel porous scaffolds of calcium phosphate cement (CPC) combined with alginate, and their potential usefulness as a three-dimensional (3-D) matrix for drug delivery and tissue engineering of bone. An α-tricalcium phosphate-based powder was mixed with sodium alginate solution and then directly injected into a fibrous structure in a Ca-containing bath. A rapid hardening reaction of the alginate with Ca(2+) helps to shape the composite into a fibrous form with diameters of hundreds of micrometers, and subsequent pressing in a mold allows the formation of 3-D porous scaffolds with different porosity levels. After transformation of the CPC into a calcium-deficient hydroxyapatite phase in simulated biological fluid the scaffold was shown to retain its mechanical stability. During the process biological proteins, such as bovine serum albumin and lysozyme, used as model proteins, were observed to be effectively loaded onto and released from the scaffolds for up to more than a month, proving the efficacy of the scaffolds as a drug delivering matrix. Mesenchymal stem cells (MSCs) were isolated from rat bone marrow and then cultured on the CPC-alginate porous scaffolds to investigate the ability to support proliferation of cells and their subsequent differentiation along the osteogenic lineage. It was shown that MSCs increasingly actively populated and also permeated into the porous network with time of culture. In particular, cells cultured within a scaffold with a relatively high porosity level showed favorable proliferation and osteogenic differentiation. An in vivo pilot study of the CPC-alginate porous scaffolds after implantation into the rat calvarium for 6 weeks revealed the formation of new bone tissue within the scaffold, closing the defect almost completely. Based on these results, the newly developed CPC-alginate porous scaffolds could be potentially useful as a 3-D matrix for drug delivery and tissue engineering of bone.     

新型大孔隙磷酸钙骨水泥作为骨组织工程支架的相关研究

目的 探讨新型大孔隙磷酸钙骨水泥(CPC)材料支架的细胞毒性和对细胞黏附、生长和增殖的影响.方法 通过添加甘露醇制孔剂和应用磷酸钠溶液作为CPC固化液的方法合成新型CPC材料.通过CCK8法检测细胞在新型CPC材料浸提液中的生长增殖情况;通过电子扫描电镜测试材料孔径和细胞在材料表面上黏附生长情况;应用力学三点弯曲实验测试新型CPC的生物力学性能.结果 新型CPC材料的孔径值达到(267.43±118.01)μm,孔隙率为(66.15±6.91)％.新型CPC材料的最大负荷、抗弯强度和坚韧度较传统CPC均增加了约1倍(P＜0.05).新型CPC材料浸提液与细胞共培养2、4、6、8d后CCK8法测试吸光度(OD)值与阴性对照组比较其差异无统计学意义(P＞0.05).结论 新型CPC材料具有强大的生物力学性能、大孔隙、高孔隙率和良好的生物相容性,有望成为理想的骨组织工程支架.     

The in vivo performance of CaP/PLGA composites with varied PLGA microsphere sizes and inorganic compositions

Enrichment of calcium phosphate (CaP) bone substitutes with poly(lactic-co-glycolic acid) (PLGA) microspheres to create porosity overcomes the problem of poor CaP degradation. The degradation of CaP–PLGA composites can be customized by changing the physical and chemical properties of PLGA and/or CaP. However, the effect of the size of dense (solid rather than hollow) PLGA microspheres in CaP has not previously been described. The present study aimed at determining the effect of different dense (i.e. solid) PLGA microsphere sizes (small (S) ～20 μm vs. large (L) ～130 μm) and of CaP composition (CaP with either anhydrous dicalcium phosphate (DCP) or calcium sulphate dihydrate (CSD)) on CaP scaffold biodegradability and subsequent bone in-growth. To this end mandibular defects in minipigs were filled with pre-set CaP–PLGA implants, with autologous bone being used as a control. After 4 weeks the autologous bone group outperformed all CaP–PLGA groups in terms of the amount of bone present at the defect site. On the other hand, at 12 weeks substantial bone formation was observed for all CaP–PLGA groups (ranging from 47 ± 25% to 62 ± 15%), showing equal amounts of bone compared with the autologous bone group (82 ± 9%), except for CaP with DCP and large PLGA microspheres (47 ± 25%). It was concluded that in the current study design the difference in PLGA microsphere size and CaP composition led to similar results with respect to scaffold degradation and subsequent bone in-growth. Further, after 12 weeks all CaP–PLGA composites proved to be effective for bone substitution.     

Synthesis and characterization of macroporous chitosan/calcium phosphate composite scaffolds for tissue engineering

Chitosan scaffolds reinforced by beta-tricalcium phosphate (beta-TCP) and calcium phosphate invert glass were fabricated with a low-cost, bioclean freeze-drying technique via thermally induced phase separation. The microstructure, mechanical performance, biodegradation, and bioactivity of the scaffolds were studied. The composite scaffolds were macroporous, and the pore structures of the scaffolds with beta-TCP and the glass appeared very different. Both the compressive modulus and yield strength of the scaffolds were greatly improved, and reinforced microstructures were achieved. The bioactivity tests showed a continuous decrease in both Ca and P concentrations of a simulated body fluid (SBF) after the scaffolds with beta-TCP were immersed in the SBF for more than 20 h, which suggests that an apatite layer might be formed on the scaffolds. However, the same was not observed for the pure chitosan scaffolds or the scaffolds incorporated with the glass. This was further confirmed by micrographs from scanning electron microscopy. This study suggests that the desirable pore structure, biodegradation rate, and bioactivity of the composite scaffolds might be achieved through controlling the ratio of chitosan and calcium phosphates or beta-TCP and the glass.