Tissue reactions of subcutaneously implanted mixture of ε-caprolactone-lactide copolymer and tricalcium phosphate. An electron microscopic evaluation in sheep

Biodegradable polymers, mainly derivates of -hydroxy acids, are widely used today in oral- and maxillofacial surgery, orthopedics, and other fields of surgery. These biomaterials are well tolerated by living tissue and fracture fixation devices made of polylactic or polyglycolic acid are clinically widely used today. Still, there are some problems in application of biodegradable polymers. Abacterial inflammatory reactions have been noticed after the clinical introduction of these devices. Both swelling and pain at the site of implantation have also been reported. The etiology of this inflammatory reaction is still unknown, despite the numerous studies. Therefore, the aim of the present study was to further characterize this inflammatory reaction in detail, by electronmicroscopy. We prepared a mixture of -caprolactone–lactide copolymer and tricalcium phosphate and placed it in the dermis in 12 sheep. Follow-up times were 9, 14, 24, and 52 weeks. We found that the mixture caused a mild inflammatory reaction. There were no signs of cell damage. Fibroblasts, macrophages, and eosinophils were found adjacent to the copolymer. The mixture is easy to handle and can be moulded into different shapes in room temperature. The results encourage us to continue our studies to develop a filling material for bone defects.     

Mixture of ε-caprolactone-lactide copolymer and tricalcium phosphate: a histological and immunohistochemical study of tissue reactions

In cranio-maxillofacial surgery, bone transplantation is needed for treatment of bony defects. An autograft, allograft or biomaterial can be used. Autogenous bone grafts are considered to be the best materials available, but there are some disadvantages in their use including donorsite morbidity, need for a second operative site and limited graft supply. A search for new bone-graft materials therefore remains necessary. We prepared a mixture of tricalcium phosphate (TCP), which is a resorbable, non-toxic, osteoconductive ceramic material and -caprolactone-lactide copolymer P(-CL/DL-LA), a resorbable polymer, and placed it in the dermis and in mandibular bone defects in 13 rabbits. Follow-up times were two, three, seven, eight, 12, 15 and 18 weeks, tissue reactions were assessed, histologically and immunohistochemically. Times of resorption of the material from tissues were reported. We found that the mixture caused a mild inflammatory reaction when placed in bone and severe inflammation when placed in dermis. No highly fluorescent layer of tenascin or fibronectin was found surrounding the implant area. The mixture was excellent to handle and very easy to place into bone defects. The results are promising and have led us to continue development of the mixture. © 1999 Kluwer Academic Publishers     

In vivo assessment of new resorbable PEG–PPG–PEG copolymer/starch bone wax in bone healing and tissue reaction of bone defect in rabbit model

In this study, in vivo performance of novel resorbable bone wax based on a miscible blend between PEG–PPG–PEG copolymer mixtures and pregelatinized starch at 0 and 25 percent by weight including hemostasis, tissue reaction and bone healing in a non-critical size tibia defect model were assessed and compared with commercial non-resorbable bone wax. Systemic reaction was evaluated by blood chemistry while local reaction, bone quantity and quality were evaluated by microcomputed tomography (microCT) and histology analyses. It was observed that the resorbable bone waxes did not show any adverse systemic reaction and resorbed from the defects within approximately 2 days after application. They were as effective as the commercial bone wax in hemostasis, but provided better adherence to the bone surface. The incorporation of pre-gelatinized starch in the formulation could further help in improved molding texture and decreased glove adherence. MicroCT and histology analyses showed that the resorbable bone waxes did not inhibit the osteogenesis whereas commercial bone wax impaired bone healing and displayed inflammation and foreign body reactions.     

The effect of rhBMP-2 and PRP delivery by biodegradable β-tricalcium phosphate scaffolds on new bone formation in a non-through rabbit cranial defect model

This study evaluated whether the combination of biodegradable β-tricalcium phosphate (β-TCP) scaffolds with recombinant human bone morphogenetic protein-2 (rhBMP-2) or platelet-rich plasma (PRP) could accelerate bone formation and increase bone height using a rabbit non-through cranial bone defect model. Four non-through cylindrical bone defects with a diameter of 8-mm were surgically created on the cranium of rabbits. β-TCP scaffolds in the presence and absence of impregnated rhBMP-2 or PRP were placed into the defects. At 8 and 16 weeks after implantation, samples were dissected and fixed for analysis by microcomputed tomography and histology. Only defects with rhBMP-2 impregnated β-TCP scaffolds showed significantly enhanced bone formation compared to non-impregnated β-TCP scaffolds (P < 0.05). Although new bone was higher than adjacent bone at 8 weeks after implantation, vertical bone augmentation was not observed at 16 weeks after implantation, probably due to scaffold resorption occurring concurrently with new bone formation.     

Study on physicochemical properties and in vitro bioactivity of tricalcium silicate–calcium carbonate composite bone cement

In this article, a novel bone cement composed of tricalcium silicate (Ca(3)SiO(5); C(3)S) and calcium carbonate (CaCO(3)) was prepared with the weight percent of CaCO(3) in the range of 0, 10, 20, 30, and 40%. The initial setting time was dramatically reduced from 90 to 45 min as the content of CaCO(3) increased from 0 to 40%, and the workable paste with a liquid/powder (L/P) ratio of 0.8 ml/g could be injected between 2 and 20 min (nozzle diameter 2.0 mm). The composite cement showed higher mechanical strength (24-27 MPa) than that of the pure Ca(3)SiO(5) paste (14-16 MPa). Furthermore, the composite cement could induce apatite formation and degrade in the phosphate buffered saline. The results indicated that the Ca(3)SiO(5)-CaCO(3) paste had better hydraulic properties than pure Ca(3)SiO(5) paste, and also the composite cement was bioactive and degradable. The novel bone cement could be a potential candidate as a bone substitute.     

Characterization and distribution of mechanically competent mineralized tissue in micropores of β-tricalcium phosphate bone substitutes

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Preparation,mechanical properties and in vitro degradability of wollastonite/tricalcium phosphate macroporous scaffolds from nanocomposite powders

A new class of scaffolds with a gain size of 200 nm was prepared from wollastonite/tricalcium phosphate (WT) nanocomposite powders (termed “nano-sintered scaffolds”) through a two-step chemical precipitation and porogen burnout techniques. For a comparison, WT scaffolds with a grain size of 2 μm were also fabricated from submicron composite powders (termed “submicron-sintered scaffolds”) under the same condition. The resultant scaffolds showed porosities between 50 ± 1.0% and 65 ± 1.0% with a pore size ranging from 100 μm to 300 μm. The WT nano-sintered scaffolds exhibited compressive strength and elastic modulus values that were about twice that of their submicron-sintered counterparts. The in vitro degradation tests demonstrated that the degradability could be regulated by the grain size of bioceramics. The decreased specific surface area of pores in the nano-sintered scaffolds led to their reduced degradation rate. The mechanical properties of the nano-sintered scaffolds exhibited less strength loss during the degradation process. The WT macroporous nano-sintered scaffolds are a promising and potential candidate for bone reconstruction applications.     

Synthesis and properties of bone cement materials in the calcium phosphate–calcium sulfate system

We have studied the influence of the cement liquid composition and the relationship between the components of the calcium sulfate–precipitated calcium phosphate system in a wide concentration range on the setting time, phase composition, microstructure, and mechanical properties of cement materials. The results demonstrate that the greatest promise is held by a magnesium phosphate-based cement liquid which, when mixed with powder, forms a high-strength phase, leading to a considerable increase in the strength of the cements. The addition of 20 wt % calcium sulfate to the starting mixture ensures dispersion hardening of the cements. We have obtained new cement materials offering a strength of up to 60 MPa, which are expected to find medical applications.     

The predominant role of collagen in the nucleation, growth, structure and orientation of bone apatite

The involvement of collagen in bone biomineralization is commonly admitted, yet its role remains unclear. Here we show that type I collagen in?vitro can initiate and orientate the growth of carbonated apatite mineral in the absence of any other vertebrate extracellular matrix molecules of calcifying tissues. We also show that the collagen matrix influences the structural characteristics on the atomic scale, and controls the size and the three-dimensional distribution of apatite at larger length scales. These results call into question recent consensus in the literature on the need for Ca-rich non-collagenous proteins for collagen mineralization to occur in vivo. Our model is based on a collagen/apatite self-assembly process that combines the ability to mimic the in vivo extracellular fluid with three major features inherent to living bone tissue, that is, high fibrillar density, monodispersed fibrils and long-range hierarchical organization.     

MBCP® biphasic calcium phosphate granules and tissucol® fibrin sealant in rabbit femoral defects: The effect of fibrin on bone ingrowth

An ageing population implies an increase in bone and dental diseases, which are in turn a source of numerous handicaps. These pathologies are an expensive burden for the European health system. As no specific bioactive materials are efficient enough to cope with this burden, we have to develop an injectable, mouldable, self-hardening bone substitute to support bone tissue reconstruction and augmentation.New, highly bioactive and suitable biomaterials have been developed to replace bone grafts in orthopedic revision and maxillofacial surgery for bone augmentation. These mouldable, self-hardening materials are based on the association of MBCP® Biphasic Calcium Phosphate Granules and Tissucol® Fibrin Sealant. The in vivo evaluation of ingrowth in relation to the composite was made in an experiment on rabbits. The results indicate that in the presence of fibrin sealant, newly-formed bone developed at a small distance from the surface of the calcium phosphate ceramic. Two different bone apposition processes were identified. Without the fibrin component (MBCP group), bone rested directly on the surface of the granules. This observation is commonly described as osteoconduction in calcium phosphate materials. On the contrary, the presence of the fibrinogen component seemed to modify this standard osteoconduction phenomenon: the newly-formed bone essentially grew at a distance from the surface of the granules, on the fibrillar network, and could be considered as an inductive phenomenon for osteogenic cell differentiation from mesenchymal stem cells.     

Calcium phosphate cement: in vitro and in vivo studies of the α-tricalcium phosphate-dicalcium phosphate dibasic-tetracalcium phosphate monoxide system

In this paper, calcium phosphate cement consisting of -tricalcium phosphate (-TCP), dicalcium phosphate dibasic (DCPD) and tetracalcium phosphate monoxide (TeCP) was investigated in vitro and in vivo. Measurements of compressive strength against soaking time in simulated body fluid (SBF) showed a rapid increase of the hardness for the first 7 days. The gained strength was retained up to 1 year and the maximal mean value was 94.7 (±14.4) MPa. X-ray diffraction (XRD) and scanning electron microscopy (SEM) presented precipitates of hydroxyapatite (HA) after mixing, also after soaking in SBF and after implantation in rat subcutaneous tissues. However, the conversion to HA happened in different ways between in vitro and in vivo exposures. Histologic examinations showed that the cement causes the same reactions at the interface with surrounding soft tissues as HA. The authors consider the cement to be a promising material as a bone substitute, bone cement or dental material, however, further studies in a paste form and in bone tissue environments are necessary.     

Correlation between properties and microstructure of laser sintered porous β-tricalcium phosphate bone scaffolds

Abstract

A porous β-tricalcium phosphate (β-TCP) bioceramic scaffold was successfully prepared with our homemade selective laser sintering system. Microstructure observation by a scanning electron microscope showed that the grains grew from 0.21 to 1.32 μm with the decrease of laser scanning speed from 250 to 50 mm min^?1. The mechanical properties increased mainly due to the improved apparent density when the laser scanning speed decreased to 150 mm min^?1. When the scanning speed was further decreased, the grain size became larger and the mechanical properties severely decreased. The highest Vickers hardness and fracture toughness of the scaffold were 3.59 GPa and 1.16 MPa m^1/2, respectively, when laser power was 11 W, spot size was 1 mm in diameter, layer thickness was 0.1–0.2 mm and laser scanning speed was 150 mm min^?1. The biocompatibility of these scaffolds was assessed in vitro with MG63 osteoblast-like cells and human bone marrow mesenchymal stem cells. The results showed that all the prepared scaffolds are suitable for cell attachment and differentiation. Moreover, the smaller the grain size, the better the cell biocompatibility. The porous scaffold with a grain size of 0.71 μm was immersed in a simulated body fluid for different days to assess the bioactivity. The surface of the scaffold was covered by a bone-like apatite layer, which indicated that the β-TCP scaffold possesses good bioactivity. These discoveries demonstrated the evolution rule between grain microstructure and the properties that give a useful reference for the fabrication of β-TCP bone scaffolds.     

Repair of goat tibial defects with bone marrow stromal cells and β-tricalcium phosphate

Tissue engineering techniques have been proven effective in bone regeneration and repairing load-bearing bone defects. Previous studies, however, have heretofore been limited to the use of slowdegradable or natural biomaterials as scaffolds. There are, however, no reports on using biodegradable, synthetic beta-tricalcium phosphate (β-TCP) as scaffolds to repair weight-bearing bone defects in large animals. In the present study, highly porous β-TCP scaffolds prepared by the polymeric sponge method were used to repair goat tibial defects. Fifteen goats were randomly assigned to one of three groups, and a 26 mm-long defect at the middle part of the right tibia in each goat was created. In Group A (six goats), a porous β-TCP ceramic cylinder that had been loaded with osteogenically induced autologous bone marrow stromal cells (BMSCs) was implanted in the defect of each animal. In Group B (six goats), the same β-TCP ceramic cylinder without any cells loaded was placed in the defect. In Group C (three goats), the defect was left untreated. In Group A, bony union can be observed by gross view, X-ray and micro-computed tomography (Micro-CT) detection, and histological observation at 32 weeks post-implantation. The implanted β-TCP scaffolds were almost completely replaced by tissue-engineered bone. Bone mineral density in the repaired area of Group A was significantly higher (p < 0.05) than that of Group B, in which scant new bone was formed in each defect and the β-TCP hadn’t been completely resorbed at 32 weeks. Moreover, the tissue-engineered bone of Group A had similar biomechanical properties as that of the normal left tibia in terms of bending strength and Young’s modulus (p > 0.05). In Group C, little or no new bone was formed, and non-union occurred, showing that the 26 mm segmental defect of the goat tibia was critical sized at 32 weeks. Thus, it can be concluded that the mechanical properties of the BMSCs/β-TCP composites could be much improved via tissue engineering approach and β-TCP might be used to repair the weight-bearing segmental defects of goat tibias. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Calcium–phosphate–silicate composite bone cement: self-setting properties and in vitro bioactivity

In this study, a novel low temperature setting calcium phosphate–silicate cement was obtained by mixing CaHPO₄ · 2H₂O (DCPD) and Ca₃SiO₅ (C₃S) with 0.75 M sodium phosphate buffers (pH = 7.0) as liquid phase. The self-setting properties of the obtained DCPD/C₃S paste with liquid to powder ratio (L/P) of 0.6 ml/g, such as setting times, injectability, degradability and compressive strength were investigated and compared with that of DCPD/CaO cement system. The results indicated that, with the weight ratio of C₃S varied from 20% to 40%, the workable DCPD/C₃S pastes could set within 20 min, and the hydrated cement showed significantly higher compressive strength (around 34.0 MPa after 24 h) than that of the DCPD/CaO cement system (approximately 10.0 MPa). Furthermore, the in vitro pH value of the cements was investigated by soaking in simulated body fluid (SBF) for 12 h, and the result indicated that the DCPD/C₃S did not induce significant increase or decrease of pH value in SBF. Additionally, the composite cement possesses better ability to support and stimulate cell proliferation than the DCPD/CaO cement. With good hydraulic properties, improved biocompatibility and moderate degradability, the novel DCPD/C₃S bone cement may be a potential candidate as bone substitute.     

Fabrication of calcium phosphate–calcium sulfate injectable bone substitute using hydroxy-propyl-methyl-cellulose and citric acid

In this study, an injectable bone substitute (IBS) consisting of citric acid, chitosan, and hydroxyl propyl methyl cellulose (HPMC) as the liquid phase and tetra calcium phosphate (TTCP), dicalcium phosphate dihydrate (DCPD) and calcium sulfate dehydrate (CSD, CaSO₄·2H₂O) powders as the solid phase, were fabricated. Two groups were classified based on the percent of citric acid in the liquid phase (20, 40 wt%). In each groups, the HPMC percentage was 0, 2, and 4 wt%. An increase in compressive strength due to changes in morphology was confirmed by scanning electron microscopy images. A good conversion rate of HAp at 20% citric acid was observed in the XRD profiles. In addition, HPMC was not obviously affected by apatite formation. However, both HPMC and citric acid increased the compressive strength of IBS. The maximum compressive strength for IBS was with 40% citric acid and 4% HPMC after 14 days of incubation in 100% humidity at 37°C.     

The static and cyclic strength of a bone–cement bond

Four-point bending static and fatigue tests were carried out on bone–cement bonds. The effects of the pressurization and the washing of the bone joint face on the bond strength were investigated. The results are summarized as follows. When the bond surface of cancellous bone is washed prior to the application of the bone cement, both the static and fatigue strengths of the bond are increased relative to the corresponding properties of unwashed bone–cement bonds. From observations of bone–cement interfaces as well as the fracture surfaces of bone–cement specimens, it has been determined that bone cement was able to infiltrate into fine holes present in washed cancellous bone. However, such infiltration occurred to a much lesser degree in the case of unwashed cancellous bone. Increasing the molding pressure during the time of cement application to the bone from 39200 to 117600 Pa had a beneficial effect on the bending strength and fatigue properties, particularly in the case of washed bone cement specimens. An increase in molding pressure also resulted in a reduction in the amount of scatter in test results.     

Observation and quantitative analysis of rat bone marrow stromal cells cultured in vitro on newly formed transparent β-tricalcium phosphate

To observe living cell morphology on ceramics by light microscopy, we fabricated a new material—transparent β - tricalcium phosphate (t-β TCP) ceramic—for the purpose of serving as a tissue culture substrate. Bone marrow stromal cells (BMSCs) were obtained from rat femora and cultured on both t-β TCP ceramic disks and culture grade polystyrene (PS) dishes in an osteogenic medium. After 1 day of culture, cell attachment and spreading on both the t-β TCP and PS substrata were equally and clearly detected by ordinary light microscopy. After 14 days of culture, extensive cell growth, alkaline phosphatase (ALP) staining, and bone mineral deposition could be detected on both substrata. In addition, quantitative biochemical analyses revealed high DNA content, ALP activity, and osteocalcin content of these cultures. This experiment is significant in that all of the results were similarly observed on both the t-β TCP and PS substrata, indicating the excellent properties of β TCP ceramics for BMSCs culture towards osteogenic differentiation.     

Novel osteoconductive β-tricalcium phosphate/poly(L-lactide-co-e-caprolactone) scaffold for bone regeneration: a study in a rabbit calvarial defect

The advantages of synthetic bone graft substitutes over autogenous bone grafts include abundant graft volume, lack of complications related to the graft harvesting, and shorter operation and recovery times for the patient. We studied a new synthetic supercritical CO₂ –processed porous composite scaffold of β-tricalcium phosphate and poly(L-lactide-co-caprolactone) copolymer as a bone graft substitute in a rabbit calvarial defect. Bilateral 12?mm diameter critical size calvarial defects were successfully created in 18 rabbits. The right defect was filled with a scaffold moistened with bone marrow aspirate, and the other was an empty control. The material was assessed for applicability during surgery. The follow-up times were 4, 12, and 24 weeks. Radiographic and micro-CT studies and histopathological analysis were used to evaluate new bone formation, tissue ingrowth, and biocompatibility. The scaffold was easy to shape and handle during the surgery, and the bone-scaffold contact was tight when visually evaluated after the implantation. The material showed good biocompatibility and its porosity enabled rapid invasion of vasculature and full thickness mesenchymal tissue ingrowth already at four weeks. By 24 weeks, full thickness bone ingrowth within the scaffold and along the dura was generally seen. In contrast, the empty defect had only a thin layer of new bone at 24 weeks. The radiodensity of the material was similar to the density of the intact bone. In conclusion, the new porous scaffold material, composed of microgranular β-TCP bound into the polymer matrix, proved to be a promising osteoconductive bone graft substitute with excellent handling properties.

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