冷冻干燥法制备的β-磷酸三钙多孔生物陶瓷支架

背景：影响冷冻干燥法制备生物陶瓷支架孔形貌和结构的因素有很多,如浆料的固含量,冷冻速率,烧结温度等。 目的：利用冷冻干燥技术结合陶瓷水基浆料,通过改变陶瓷浆料组成,制备不同形貌和孔隙率的β-磷酸三钙多孔生物陶瓷支架,并分析其影响因素。 方法：制备不同固含量和不同聚乙烯醇含量的β-磷酸三钙浆料,经冷冻、干燥及高温烧结得到多孔生物陶瓷支架。应用X射线衍射技术进行物相分析;扫描电镜观察支架截面的微观结构;阿基米德排水法测量支架材料的孔隙率。 结果与结论：X射线衍射图谱显示制备的支架材料各衍射峰强度与位置与β-磷酸三钙标准衍射谱吻合良好;浆料未添加聚乙烯醇时,多孔支架的孔径和孔隙率随着浆料固含量的增加而减小,支架由大的柱状孔和多孔的陶瓷壁组成;当浆料加入聚乙烯醇后,制备的支架由柱状孔转变为三维连通的网状孔,且随聚乙烯醇含量的增加材料的孔隙率升高。说明利用冷冻干燥法,通过控制陶瓷浆料的组成,可制备出形貌和孔隙率可控的生物陶瓷支架。     

Bone substitute: transforming beta-tricalcium phosphate porous scaffolds into monetite

The goal of the present study was to assess the possibility to change the composition of a calcium phosphate scaffold from a high-temperature phase to a phase only stable at or close to room temperature without macrostructural changes. For that purpose, macroporous beta-TCP scaffolds were converted into alpha-TCP by high-temperature thermal treatment and then dipped into a phosphoric acid solution to obtain a more acidic calcium phosphate phase called monetite or dicalcium phosphate (DCP; CaHPO4). Two different solid-to-liquid ratios (SLR: 0.067 and 0.200g/mL) and three different temperatures (T: 37, 60 and 80 degrees C) were used. The reaction was followed by measuring the change of sample size and weight, by determining the compositional changes by X-ray diffraction (Rietveld analysis), and by looking at the micro- and macrostructural changes by scanning electron microscopy and micro-computed tomography. The results revealed that the transformation proceeded faster at a higher temperature and a higher SLR value but was achieved within a few days in all cases. Morphologically, the porosity decreased by 10%, the pore size distribution became wider and the mean macro pore size was reduced from 0.28 to 0.19mm. The fastest conversion and the highest compressive strength (9MPa) were measured using an incubation temperature of 80 degrees C and an SLR value of 0.2g/mL.     

How to optimize seeding and culturing of human osteoblast-like cells on various biomaterials

The optimization of seeding and culturing of human osteoblast-like cells on three collagen-based biomaterials (bovine, equine and calf collagen membrane) was studied by cell proliferation and cell colonization (scanning electron microscopy) analysis. Osteoblasts of five patients were seeded onto the three biomaterials and two different parameters were varied: the time intervals between initial seeding and adding culture medium (2 h 6 h. 12 h, 24 h) and the seeding concentration (1 x 10(5), 1 x 10(6), 2 x 10(6)cells/ml) of cells onto biomaterials. The results of the study demonstrated that the time interval between seeding osteoblasts and adding culture medium as well as the seeding concentration effects the cell proliferation and the cell colonization. The best proliferation rate was achieved by adding the culture medium 2 h after initial seeding and with a seeding density of 1 x 10(5) cells/ml. Moreover, all three biomaterials resulted in different proliferation rates. The best proliferation rate resulted with the bovine collagen membrane. In conclusion, the examined parameters are very important for the development of the tissue engineering techniques and in a larger perspective also for reconstructive surgery.     

Synthesis and characterization of porous beta-tricalcium phosphate blocks

Porous beta-tricalcium phosphate (beta-TCP) blocks with four different macropore sizes (pore larger than 50 microm were synthesized using "calcium phosphate emulsions", and characterized by optical, geometrical, gravimetric, and radiological methods. The reproducibility of the synthesis method was excellent. Moreover, the macropore size could be easily controlled without modifying the microporosity (pore smaller than 50 microm) or the total porosity (microporosity+macroporosity). Based on the initial composition of the blocks and their final apparent density, the microporosity, macroporosity, and the total block porosity were calculated to be close to 21%, 54%, and 75%, respectively. These values were confirmed by microcomputed tomography (microCT). The mean macropore diameters were close to 150, 260, 510 and 1220 microm, as measured optically. Consistently lower values (25% lower) were obtained by microCT, but the linear correlation between microCT and optical method was high (r(2)>0.97). The macropore size distribution calculated from microCT scans appears to be narrow and normally distributed. The very good correlation between the results of the various methods and the possibility to determine the pore size distribution suggest that microCT is an ideal tool to non-destructively characterize macroporous calcium phosphate bone substitutes.     

A new bone-inducing biodegradable porous beta-tricalcium phosphate

A new type of degradable biomaterial with bone-inducing capacity was made by combining porous beta-tricalcium phosphate (beta-TCP) with a delivery system for recombinant human bone morphogenetic protein-2 (rhBMP-2). The BMP delivery system consisted of a block copolymer composed of poly-D,L-lactic acid with random insertion of p-dioxanone and polyethylene glycol (PLA-DX-PEG), a known biocompatible and biodegradable material. The efficacy of this biomaterial in terms of its bone-inducing capacity was examined by ectopic bone formation in the dorsal muscles of the mouse. In the beta-TCP implants coated with the PLA-DX-PEG polymer containing more than 0.0025% (w/w) of rhBMP-2, new ectopic bone tissues with marrow were consistently found on the surface of implants. The radiographic density of beta-TCP was diminished in a time-dependent manner. On histological examination, numerous multinucleated osteoclasts with positive tartrate-resistant acid-phosphatase (TRAP) staining were noted on the surface of the beta-TCP. These experimental results indicate that beta-TCP implants coated with synthetic rhBMP-2 delivery system might provide effective artificial bone-graft substitutes with osteoinductive capacity and biodegradable properties. In addition, this type of biomaterial may require less rhBMP-2 to induce significant new bone mass.     

In vitro degradation and fracture toughness of multilayered porous poly(propylene fumarate)/beta-tricalcium phosphate scaffolds

This study investigated the in vitro degradation of poly(propylene fumarate)/beta-tricalcium phosphate (PPF/beta-TCP) scaffolds in pH 7.4 phosphate-buffered saline at 37 degrees C. Scaffold design consisted of three layers: two solid layers about a central layer of porous PPF foam. Solid PPF with molecular weights of 810 and 1450 Da was crosslinked under UV light. PPF foam was prepared by a photocrosslinking, porogen-leaching method with an initial porogen content of 80 wt % and two sizes, 150-300 and 300-500 microm. Comparison of initial and residual weights demonstrated a 14.3 +/- 2.0% loss of mass at 3 weeks and a 16.6 +/- 1.8% loss of mass at 6 weeks. Observed pH values for all constructs remained stable (7.15-7.40) throughout the 3 to 6 weeks. Scanning electron micrographs of these scaffolds revealed some loss of foam material between 3 and 6 weeks; however, foam microarchitecture was intact. Solid PPF fracture toughness was tested for high and low molecular weight PPF, 0.376 +/- 0.004 and 0.134 +/- 0.015 MPa(m)1/2, respectively. These values are roughly one magnitude less than human cortical bone.     

Sintering and robocasting of beta-tricalcium phosphate scaffolds for orthopaedic applications

beta-Tricalcium phosphate (beta-TCP) scaffolds with designed, three-dimensional (3-D) geometry and mesoscale porosity have been fabricated by direct-write assembly (robocasting) techniques. Concentrated beta-TCP inks with suitable viscoelastic properties were developed to enable the fabrication of the complex 3-D structures. A comprehensive study of the sintering behavior of TCP as a function of the calcium content in the starting powder was also carried out, and the optimal heat treatment for fabricating scaffolds with dense beta-TCP rods has been determined. Such analysis provides clues to controlling the microstructure of the fabricated structures and, therefore, enabling the fabrication by robocasting of TCP scaffolds with tailored performance for bone tissue engineering applications.     

Effects of the architecture of tissue engineering scaffolds on cell seeding and culturing

The advance of rapid prototyping techniques has significantly improved control over the pore network architecture of tissue engineering scaffolds. In this work, we have assessed the influence of scaffold pore architecture on cell seeding and static culturing, by comparing a computer designed gyroid architecture fabricated by stereolithography with a random pore architecture resulting from salt leaching. The scaffold types showed comparable porosity and pore size values, but the gyroid type showed a more than 10-fold higher permeability due to the absence of size-limiting pore interconnections. The higher permeability significantly improved the wetting properties of the hydrophobic scaffolds and increased the settling speed of cells upon static seeding of immortalised mesenchymal stem cells. After dynamic seeding followed by 5 days of static culture gyroid scaffolds showed large cell populations in the centre of the scaffold, while salt-leached scaffolds were covered with a cell sheet on the outside and no cells were found in the scaffold centre. It was shown that interconnectivity of the pores and permeability of the scaffold prolonged the time of static culture before overgrowth of cells at the scaffold periphery occurred. Furthermore, novel scaffold designs are proposed to further improve the transport of oxygen and nutrients throughout the scaffolds and to create tissue engineering grafts with a designed, pre-fabricated vasculature.     

Centrifugal seeding increases seeding efficiency and cellular distribution of bone marrow stromal cells in porous biodegradable scaffolds

Bone marrow stromal cells (MSCs) are a promising cell source for a variety of tissue engineering applications, given their ready availability and ability to differentiate into multiple cell lineages. MSCs have been successfully used to create neotissue for cardiovascular, urological, and orthopedic reconstructive surgical procedures in preclinical studies. The ability to optimize seeding techniques of MSCs onto tissue engineering scaffolds and the ability to control neotissue formation in vitro will be important for the rational design of future tissue engineering applications using MSCs. In this study we investigated the effect of centrifugal force on seeding MSCs into a biodegradable polyester scaffold. MSCs were isolated and seeded onto porous scaffold sections composed of nonwoven polyglycolic acid mesh coated with poly(L-lactide-co-epsilon-caprolactone). Compared to standard static seeding techniques, centrifugal seeding increased the seeding efficiency by 38% (p < 0.007) and significantly improved cellular distribution throughout the scaffold. Overall, centrifugal seeding of MSCs enhances seeding efficiency and improves cellular penetration into scaffolds, making it a potentially useful technique for manipulating neotissue formation by MSCs for tissue engineering applications.     

Antibiotic-loaded poly-epsilon-caprolactone and porous beta-tricalcium phosphate composite for treating osteomyelitis

A composite of poly-epsilon-caprolactone (PCL) loaded with gatifloxacine (GFLX), an antibiotic, and a beta-tricalcium phosphate (betaTCP) porous ceramic body was prepared by a solvent-free process in which no toxic solvent was used. GFLX mostly retained its bactericidal property after the processing. The composite of GFLX-loaded PCL and betaTCP ceramic released GFLX for 4 weeks in Hanks' balanced solution, and had sustained bactericidal activity against Streptococcus milleri and Bacteroides fragilis for at least 1 week. The composite of the GFLX-loaded PCL and betaTCP ceramic was implanted in an osteomyelitis lesion induced by S. milleri and B. fragilis in the rabbit mandible. The osteomyelitis lesion expanded in the mesial-distal direction when no composite was implanted or when the lesion was treated with debridement only. The composite of GFLX-loaded PCL and betaTCP showed efficacy in controlling infection at the bone defect formed by debridement, and supported bone tissue reconstruction at the bone defect. Twelve and 50 weeks after the implantation, the inflammation even disappeared. New bone formation was observed on the surface of the composite after 4 weeks. After 50 weeks, ingrowth of bone tissues with vascular channels was observed along the PCL and betaTCP interface, which indicated degradation of PCL and/or betaTCP ceramic at the ceramic/polymer interface followed by replacement by bone tissues. The GFLX concentrations in the serum and soft tissues were very low. Therefore, the composite of GFLX-loaded PCL and betaTCP ceramic would help arrest osteomyelitis when it is used in addition to intravenous antibiotic administration, and help new bone formation and osteoconduction.     

In vivo response of porous hydroxyapatite and beta-tricalcium phosphate prepared by aqueous solution combustion method and comparison with bioglass scaffolds

Pure hydroxyapatite (HAp) and a biphasic calcium phosphate [containing 90% of beta-tri-calcium phosphate (beta-TCP) and 10% HAp] were tailored through an aqueous solution combustion synthesis. Porous struts were prepared using all the powders along with bioglass, a known bioactive material, and subsequently characterized. Sterilized struts were implanted to the lateral side of radius bone of 24 black Bengal goats of either sex, in which a blank hole was left unfilled in a group of six specimens to act as control. The bone formation response of the three implanting materials in vivo has been studied using scanning electron microscope and histological analysis in contrast with positive controls. Push-out tests were used to assess the mechanical strength at the bone-biomaterial interface. It was observed that interfacial response was strongly dependent on combinations of different physical and chemical parameters. The surface of beta-TCP exhibited similar characteristics of bone and was distinct from those of intervening apatite layer of bioglass. Lower bone ingrowth and reduced strength was observed with HAp compared to beta-TCP/bioglass-based implants. Bone formation response of the Ca-P material varied according to the composition of the implanting material, which could be tailored through this novel synthesis.     

Repair of canine mandibular bone defects with bone marrow stromal cells and porous beta-tricalcium phosphate

Tissue engineering has become a new approach for repairing bone defects. Previous studies have been limited to the use of slow-degradable scaffolds with bone marrow stromal cells (BMSCs) in mandibular reconstruction. In this study, a 30 mm long mandibular segmental defect was repaired by engineered bone graft using osteogenically induced autologous BMSCs seeded on porous beta-tricalcium phosphate (beta-TCP, n=5). The repair of defects was compared with those treated with beta-TCP alone (n=6) or with autologous mandibular segment (n=4). In the BMSCs/beta-TCP group, new bone formation was observed from 4 weeks post-operation, and bony-union was achieved after 32 weeks, which was detected by radiographic and histological examination. In contrast, minimal bone formation with almost fibrous connection was observed in the group treated with beta-TCP alone. More importantly, the engineered bone with BMSCs/beta-TCP achieved a satisfactory biomechanical property in terms of bending load strength, bending displacement, bending stress and Young's modulus at 32 weeks post-operation, which was very close to those of contralateral edentulous mandible and autograft bone (p>0.05). Based on these results, we conclude that engineered bone from osteogenically induced BMSCs and biodegradable beta-TCP can well repair the critical-sized segmental mandibular defects in canines.     

Flow perfusion culture of human mesenchymal stem cells on silicate-substituted tricalcium phosphate scaffolds

Autologous bone grafts are currently the gold standard for treatment of large bone defects, but their availability is limited due to donor site morbidity. Different substitutes have been suggested to replace these grafts, and this study presents a bone tissue engineered alternative using silicate-substituted tricalcium phosphate (Si-TCP) scaffolds seeded with human bone marrow-derived mesenchymal stem cells (hMSC). The cells were seeded onto the scaffolds and cultured either statically or in a perfusion bioreactor for up to 21 days and assessed for osteogenic differentiation by alkaline phosphatase activity assays and by quantitative real-time RT-PCR on bone markers. During culture, cells from the flow cultured constructs demonstrated improved proliferation and osteogenic differentiation verified by a more pronounced expression of several bone markers, e.g. alkaline phosphatase, osteopontin, Runx2, bone sialoprotein II, and bone morphogenetic protein 2. Cells and matrix were distributed homogeneously throughout the entire scaffold in flow culture, whereas only a peripheral layer was obtained after static culture. A viable and homogenous ex vivo bone construct with superior osteogenic properties was produced in dynamic culture and may provide a replacement for autologous grafts.     

Growth of osteoblast-like cells on biomimetic apatite-coated chitosan scaffolds

Porous scaffold materials that can provide a framework for the cells to adhere, proliferate, and create extracellular matrix are considered to be suitable materials for bone regeneration. Interconnected porous chitosan scaffolds were prepared by freeze-drying method, and were mineralized by calcium and phosphate solution by double-diffusion method to form nanoapatite in chitosan matrix. The mineralized chitosan scaffold contains hydroxyapatite nanocrystals on the surface and also within the pore channels of the scaffold. To assess the effect of apatite and porosity of the scaffolds on cells, human osteoblast (SaOS-2) cells were cultured on unmineralized and mineralized chitosan scaffolds. The cell growth on the mineralized scaffolds and on the pure chitosan scaffold shows a similar growth trend. The total protein content and alkaline phosphatase enzyme activity of the cells grown on scaffolds were quantified, and were found to increase over time in mineralized scaffold after 1 and 3 weeks of culture. The electron microscopy of the cell-seeded scaffolds showed that most of the outer macropores became sealed off by a continuous layer of cells. The cells spanned around the pore wall and formed extra cellular matrix, consisting mainly of collagen in mineralized scaffolds. The hydroxyproline content also confirmed the formation of the collagen matrix by cells in mineralized scaffolds. This study demonstrated that the presence of apatite nanocrystals in chitosan scaffolds does not significantly influence the growth of cells, but does induce the formation of extracellular matrix and therefore has the potential to serve for bone tissue engineering.     

Flow perfusion culture of human fetal bone cells in large beta-tricalcium phosphate scaffold with controlled architecture

One unsolved problem in bone tissue engineering is how to enable the survival and proliferation of osteoblastic cells in large scaffolds. In this work, large beta-tricalcium phosphate scaffolds with tightly controlled channel architectures were fabricated and a custom-designed perfusion bioreactor was developed. Human fetal bone cells in third passage were seeded onto the scaffolds and cultured in static or flow perfusion conditions for up to 16 days. Compared with nonperfused constructs, flow perfused constructs demonstrated improved cells proliferation and differentiation according to cell viability, glucose consumption, alkaline phosphatase activity, and osteopontin. Moreover, after 16 days of perfusion culture, a homogenous layer composed of cells and mineralized matrix throughout the whole scaffold was observed by scanning electron microscopy and histological study. In contrast, cells were located only along the scaffold perimeter in static culture. These results demonstrated the feasibility and benefit of perfusion culture in conjunction with well-defined three-dimensional environment for large bone graft construction. Porous scaffold with controlled architecture can be a potential tool to evaluate the effects of scaffold specific geometry on fluid flow configuration and cell behavior under perfusion culture.     

Osteogenic and angiogenic potentials of monocultured and co-cultured human-bone-marrow-derived mesenchymal stem cells and human-umbilical-vein endothelial cells on three-dimensional porous beta-tricalcium phosphate scaffold

The use of biodegradable beta-tricalcium phosphate (β-TCP) scaffolds holds great promise for bone tissue engineering. However, the effects of β-TCP on bone and endothelial cells are not fully understood. This study aimed to investigate cell proliferation and differentiation of mono- or co-cultured human-bone-marrow-derived mesenchymal stem cells (hBMSCs) and human-umbilical-vein endothelial cells (HUVECs) on a three-dimensional porous, biodegradable β-TCP scaffold. In co-culture studies, the ratios of hBMSCs:HUVECs were 5:1, 1:1 and 1:5. Cellular morphologies of HUVECs, hBMSCs and co-cultured HUVECs/hBMSCs on the β-TCP scaffolds were monitored using confocal and scanning electron microscopy. Cell proliferation was monitored by measuring the amount of double-stranded DNA (dsDNA) whereas hBMSC and HUVEC differentiation was assessed using the osteogenic and angiogenic markers, alkaline phosphatase (ALP) and PECAM-1 (CD31), respectively. Results show that HUVECs, hBMSCs and hBMSCs/HUVECs adhered to and proliferated well on the β-TCP scaffolds. In monoculture, hBMSCs grew faster than HUVECs on the β-TCP scaffolds after 7 days, but HUVECs reached similar levels of proliferation after 14 days. In monoculture, β-TCP scaffolds promoted ALP activities of both hBMSCs and HUVECs when compared to those grown on tissue culture well plates. ALP activity of cells in co-culture was higher than that of hBMSCs in monoculture. Real-time polymerase chain reaction results indicate that runx2 and alp gene expression in monocultured hBMSCs remained unchanged at days 7 and 14, but alp gene expression was significantly increased in hBMSC co-cultures when the contribution of individual cell types was not distinguished.     

Preparation and characterization of macroporous chitosan-gelatin/beta-tricalcium phosphate composite scaffolds for bone tissue engineering

A biodegradable composite scaffold was developed using beta-tricalcium phosphate (beta-TCP) with chitosan (CS) and gelatin (Gel) in the form of a hybrid polymer network (HPN) via co-crosslinking with glutaraldehyde. Various types of scaffolds were prepared by freezing and lyophilizing. These scaffolds were characterized by Fourier transform infrared, X-ray diffractometer, and scanning electron microscopy. The macroporous composite scaffolds exhibited different pore structures. Compressive properties were improved, especially compressive modulus from 3.9-10.9 MPa. Biocompatibility was evaluated subcutaneously on rabbits. A mild inflammatory response was observed over 12 weeks. The results suggest that the scaffolds can be utilized in nonloading bone regeneration.     

Engineering of a dermal equivalent: seeding and culturing fibroblasts in PEGT/PBT copolymer scaffolds

The engineering of dermal skin substitutes, using autologous fibroblasts, requires high seeding efficiencies, a homogeneous cell distribution in the scaffolds, and optimal culture conditions. Dynamic seeding in spinner flasks was used to seed and subsequently culture fibroblasts in three-dimensional scaffolds. Several seeding and culture variables were investigated. Simulation of medium movement with microspheres showed that three different regions existed in medium (outer, middle, and inner), where overall particle movement was different. In the middle region the flow was turbulent and scaffolds were best placed in this region. After fibroblast seeding, methylene blue staining and scanning electron microscopy analysis of the scaffolds showed that at a low stirring speed (20 rpm) fibroblasts attached mainly onto the upper part of the scaffold, and at 40 and 60 rpm fibroblasts attached and spread throughout the scaffolds. Measurements of total DNA content per scaffold showed that lower stirring speeds (20 and 40 rpm) resulted in significantly higher cell-seeding efficiencies (20 rpm, 99.8 +/- 11.3%; 40 rpm, 93.8 +/- 10.5%) compared with 60 rpm (85.9 +/- 5.3%). Seeding kinetics were comparable for all three speeds investigated. In subsequent studies, 40 rpm was chosen for seeding. Using initial cell numbers ranging from 0.3 x 10(6) to 1.5 x 10(6) fibroblasts per scaffold, seeding efficiencies higher than 85% were consistently found (n = 4). The culture of fibroblast-seeded scaffolds at different stirring speeds (10-80 rpm) showed that stirring speeds higher than 10 rpm significantly stimulated fibroblast proliferation and glycosaminoglycan and collagen deposition as compared with 10 rpm. After 21 days, scaffolds cultured at 80 rpm showed significantly more collagen deposition as compared with those maintained at lower speeds. In conclusion, to achieve high seeding efficiencies, uniform fibroblast distribution and tissue formation in a three-dimensional scaffold, fibroblasts can be dynamically seeded at 40 rpm and subsequently cultured at a stirring speed of 60-80 rpm in spinner flasks. This flexible system shows that it is feasible to tissue engineer autologous dermal substitutes in a clinically acceptable time frame.     

A 1-min method for homogenous cell seeding in porous scaffolds

The aim of this study was to develop and evaluate a simple and rapid cell seeding procedure for both calcium phosphate ceramic scaffolds and polymer scaffolds. Poly(d,l-lactic acid) and β-tri-calcium phosphate scaffolds were seeded with MC3T3-E1 cells in a syringe. Scaffolds were put in the syringe. After replacing the plunger, the cell suspension was drawn into the syringe. The syringe was closed and the plunger was retracted to the volume of the cell suspension to create a vacuum. This was done for 3?×?10?s. By this procedure, cells were homogenously distributed throughout the scaffold. The efficiency of cell seeding was approximately 60% for both scaffolds independent of the initial cell density. The hypotension the cells experienced for 3?×?10?s did not affect the proliferation capacity of the cells. In conclusion, this method of syringe-vacuum cell seeding is easy, quick, cheap, and easily to perform at an operating theatre.