The association of hydrogel and biphasic calcium phosphate in the treatment of dehiscence-type peri-implant defects: an experimental study in dogs

Hydrogel polymers have many applications in regenerative medicine. The aim of this study in dogs was to investigate bone regeneration in dehiscence-type peri-implant defects created surgically and treated with (i) biphasic calcium phosphate (BCP) granules alone; (ii) a composite putty hydroxypropyl methylcellulose (HPMC)/BCP (MBCP/putty); and (iii) a polymer crosslinked membrane of silanized-HPMC (Si-HPMC/BCP) compared with empty controls. At 3 months, new bone formation was significantly more important in defects filled with HPMC/BCP or Si-HPMC/BCP compared with spontaneous healing in control (P = 0.032 and P = 0.046 respectively) and more substantial compared with BCP alone. Furthermore, new bone formation in direct contact with the implant surface was observed in all three groups treated with BCP. The addition of HPMC to the BCP granules may have enhanced the initial stability of the material within the blood clot in these large and complex osseous defects. The Si-HPMC hydrogel may also act as an occlusive membrane covering the BCP, which could improve the stability of the granules in the defect area. However, the crosslinking time of the Si-HPMC is too long for easy handling and the mechanical properties remain to be improved. The composite MBCP/putty appears to be a valuable bone-graft material in complex defects in periodontology and implantology. These encouraging results should now be confirmed in clinical studies.     

Bone regeneration using an injectable calcium phosphate/autologous iliac crest bone composites for segmental ulnar defects in rabbits

Background Treatment of segmental bone loss remains a challenge in skeletal repair. A major therapeutic goal is the development of implantable materials that will promote bone regeneration. Objective We evaluate bone regeneration in grafts containing different concentrations autologous iliac crest bone (ACB) particles, carried in a new injectable calcium phosphate cement (CPC), in ulnar bone defects in rabbits. Methods Large upper-mid-diaphyseal defects (10 mm) were created in the left ulnae of 60 skeletally mature New Zealand white rabbits. ACB concentrations of 0, 25, 50, 75, and 100% (by volume) in CPC were used to fill operated sites. Defect bridging was monitored by serial radiography at 4, 8, and 12 weeks post-operation. Samples were then examined histologically and by manual palpation to determine the extent of new bone formation. Results At 4 weeks, we observed more elaborate structures and extensive absorption in ulnae treated with mixtures containing low concentrations of ACB (such as 0% and 25% volume of ACB/CPC), compared with those treated with mixtures containing high concentrations of ACB (such as 75% and 100% volume of ACB/CPC). At 8 weeks, histomorphometry revealed increased trabecular area and volume in the group treated with high ACB concentrations compared with those treated with low ACB concentrations. At 12 weeks, complete cortical bridging and regeneration of marrow space were detected in groups treated with high concentrations of ACB, and the amount of new bone regeneration was greater in these groups than in those treated with low ACB concentrations. Conclusions Treatment of rabbit ulnar defects with injectable CPC carrying an optimized concentration of ACB particles can lead to cortical bridging and bone marrow regeneration within 12 weeks.     

Developments in injectable multiphasic biomaterials. The performance of microporous biphasic calcium phosphate granules and hydrogels

Calcium phosphate bioceramic granules associated with hydrosoluble polymers were developed as bone substitutes for various maxillofacial and orthopaedic applications. These injectable bone substitutes, support and regenerate bone tissue and resorb after implantation. The efficiency of these multiphasic materials is due to the osteogenic and osteoconductive properties of the microporous biphasic calcium phosphate. The associated hydrosoluble polymers are considered as carriers in order to achieve the rheological properties of injectable bone substitutes (IBS). In this study, we used 2 semi synthetic hydrosoluble polymers of polysaccharidic origin. The hydroxy propyl methyl cellulose (HPMC), with and without silane, was combined with microporous BCP granules. The presence of silane induced considerable gelation of the suspension. The 2 IBS used (without gelation, IBS1, with gelation, IBS2) were implanted in critical size femoral epiphysis defects in rabbits. No foreign body reactions were observed in either sample. However, because of the higher density from gelation, cell colonisation followed by bone tissue ingrowth was delayed over time with IBS2 compared to the IBS1 without gelation. The results showed resorption of the BCP granule and bone ingrowth at the expense of both IBS with different kinetics. This study demonstrates that the hydrogel cannot be considered merely as a carrier. The gelation process delayed cell and tissue colonisation by slow degradation of the HPMC Si, compared to the faster release of HPMC with IBS1, in turn inducing faster permeability and spaces for tissue ingrowth between the BCP granules.     

The stability mechanisms of an injectable calcium phosphate ceramic suspension

Calcium phosphate ceramics are widely used as bone substitutes in dentistry and orthopedic applications. For minimally invasive surgery an injectable calcium phosphate ceramic suspension (ICPCS) was developed. It consists in a biopolymer (hydroxypropylmethylcellulose: HPMC) as matrix and bioactive calcium phosphate ceramics (biphasic calcium phosphate: BCP) as fillers. The stability of the suspension is essential to this generation of “ready to use” injectable biomaterial. But, during storage, the particles settle down. The engineering sciences have long been interested in models describing the settling (or sedimentation) of particles in viscous fluids. Our work is dedicated to the comprehension of the effect of the formulation on the stability of calcium phosphate suspension before and after steam sterilization. The rheological characterization revealed the macromolecular behavior of the suspending medium. The investigations of settling kinetics showed the influence of the BCP particle size and the HPMC concentration on the settling velocity and sediment compactness before and after sterilization. To decrease the sedimentation process, the granule size has to be smaller and the polymer concentration has to increase. A much lower sedimentation velocity, as compared to Stokes law, is observed and interpreted in terms of interactions between the polymer network in solution and the particles. This experimentation highlights the granules spacer property of hydrophilic macromolecules that is a key issue for interconnection control, one of the better ways to improve osteoconduction and bioactivity.     

Periodontal regeneration in experimentally-induced alveolar bone dehiscence by an improved porous biphasic calcium phosphate ceramic in beagle dogs

Regeneration of lost periodontium is the focus of periodontal therapy. To achieve the effective regeneration, a number of bone graft substitute materials have been developed. This study aimed to investigate the histological response in alveolar bone dehiscences which were filled with an improved biphasic calcium phosphate (BCP) ceramic with more reasonable pore diameter, pore wall thickness and porosity. Twenty-four alveolar bone dehiscences were made surgically in twelve beagle dogs by reflecting mucoperiosteal flaps on the buccal aspect of bilateral lower second premolars and removing alveolar bone. The left dehiscences were treated with BCP ceramic and the contralaterals were cured with the open flap debridement (OFD) as controls. Three dogs were used at week 4, 12, and 24 respectively. Histological observations were processed through three-dimensional micro-computed tomographic imaging, fluorescence and light microscopy. The histological study indicated that the biphasic ceramic was biocompatible, and regeneration was achieved more effectively through the BCP treatment. There were also arrest of epithelial migration apically and formation of new bone and cementum, as well as proliferation of fibrous connective tissues that became attached to the newly formed cementum at week 24, while there was no significant periodontal regeneration in the OFD group only with epithelial tissue migrating into the dehiscence regions. Clinically speaking, though the surgical location formed a limitation to the application of the improved BCP on the periodontal regeneration, the actual result was positive. It proved that the BCP had biocompatibility and was able to act as a stable scaffold to induce periodontal regeneration effectively.     

一种可注射可降解磷酸钙骨水泥的结构与性能

通过采用部分结晶磷酸钙和磷酸氢钙制备了新型可注射可降解磷酸钙骨水泥.研究表明:该材料具备优良的可注射性能,并通过添加变性淀粉,显著改善了材料的抗溃散性能,骨水泥的水化产物是直径和长度在分别约为100和1000nm左右的棒状类骨羟基磷灰石.所研制的骨水泥在体温(37℃)条件下凝结较快,而在室温(25℃)和冷藏温度(5℃)可在较长时间保持不固化,这就为骨水泥的临床应用提供了很有利的条件.体外溶血试验、体外细胞毒试验、热原性试验、小鼠的急性毒性试验、微核试验、豚鼠的致敏性试验、小鼠的肌内埋植试验及兔的骨内埋植试验等一系列毒性及生物相容性试验表明该材料无毒副作用,具有良好的生物相容性.复合rhBMP-2的可注射磷酸钙骨水泥植入猕猴椎体后的近远期影像学和组织学观察表明,骨水泥可降解且降解和新骨长入基本同步.     

Cytocompatibility evaluation of microwave sintered biphasic calcium phosphate scaffolds synthesized using pH control

Compounds belonging to the calcium phosphate (CaP) system are known to be major constituents of bone and are bioactive to different extents in vitro and in vivo. Their chemical similarity makes them prime candidates for implants and bone tissue engineering scaffolds. CaP nanoparticles of amorphous hydroxyapatite (aHA) and dicalcium phosphate dihydrate (DCPD) were synthesized using chemical precipitation. Uniaxially pressed aHA and DCPD powders were subjected to microwave radiation to promote solid state phase transformations resulting in crystalline hydroxyapatite (HA), tricalcium phosphate (TCP) and biphasic compositions: HA/TCP and TCP/calcium pyrophosphate (CPP) and their subsequent densification. Phase composition of microwave sintered compacts was confirmed via X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). Solution pH during crystal growth was found to have a profound effect on particle morphology and post-sintered phases, despite constant sintering temperature.Cytocompatibility assessment using 7F2 cells, corresponding to adult mouse osteoblasts, on microwave and conventional, furnace sintered samples demonstrated that manufacturing method does not impact cellular viability after 24 h or proliferation over 7 days. New CaP deposition and extracellular matrix components were observed in vitro via scanning electron microscopy (SEM).     

Optimisation of the mechanical and handling properties of an injectable calcium phosphate cement

Calcium phosphate cements have the potential to be successful in minimally invasive surgical techniques, like that of vertebroplasty, due to their ability to be injected into a specific bone cavity. These bone cements set to produce a material similar to that of the natural mineral component in bone. Due to the ceramic nature of these materials they are highly brittle and it has been found that they are difficult to inject. This study was carried out to determine the factors that have the greatest effect on the mechanical and handling properties of an apatitic calcium phosphate cement with the use of a Design of Experiments (DoE) approach. The properties of the cement were predominantly influenced by the liquid:powder ratio and weight percent of di-sodium hydrogen phosphate within the liquid phase. An optimum cement composition was hypothesised and tested. The mechanical properties of the optimised cement were within the clinical range for vertebroplasty, however, the handling properties still require improvement.     

The thermal stability of hydroxyapatite in biphasic calcium phosphate ceramics

Biphasic calcium phosphate ceramics (BCP) comprising a mix of non-resorbable hydroxyapatite (HA) and resorbable β-tricalcium phosphate (β-TCP) are particularly suitable materials for synthetic bone substitute applications. In this study, HA synthesised by solid state reaction was mechanically mixed with β-TCP, then sintered to form a suite of BCP materials with a wide range of HA/β-TCP phase content ratios. The influence of sintering temperature and composition on the HA thermal stability was quantified by X-ray diffraction (XRD). The pre-sinter β-TCP content was found to strongly affect the post-sinter HA/β-TCP ratio by promoting the thermal decomposition of HA to β-TCP, even at sintering temperatures as low as 850 °C. For BCP material with pre-sinter HA/β-TCP = 40/60 wt%, approximately 80% of the HA decomposed to β-TCP during sintering at 1000 °C. Furthermore, the HA content appeared to influence the reverse transformation of α-TCP to β-TCP expected upon gradual cooling from sintering temperatures greater than 1125 °C. Because the HA/β-TCP ratio dominantly determines the rate and extent of BCP resorption in vivo, the possible thermal decomposition of HA during BCP synthesis must be considered, particularly if high temperature treatments are involved.     

Bone tissue formation in sheep muscles induced by a biphasic calcium phosphate ceramic and fibrin glue composite

Some biomaterials are able to induce ectopic bone formation in muscles of large animals. The osteoinductive potential of macro- micro-porous biphasic calcium phosphate (MBCP) ceramic granules with fibrin glue was evaluated by intramuscular implantation for 6 months in six adult female sheep. The MBCP granules were 1–2 mm in size and were composed of hydroxyapatite (HA) and beta-tricalcium phosphate (β-TCP) in a 60/40 ratio. The fibrin glue was composed of fibrinogen, thrombin and other biological factors. After 6 months of implantation in the dorsal muscles of sheep, the explants were rigid. Histology, back-scattered electron microscopy and micro-computed tomography of the implants indicated that approximately 12% of mineralized bone had formed in between the MBCP granules. The ectopic bone appeared well-mineralized with mature osteocytes and Haversian structures. In addition, the number and thickness of bone trabeculae formed in between the MBCP particles were similar to those measured in trabecular bone in sheep. The overall results therefore confirmed the formation of well-mineralized ectopic bone tissue after intramuscular implantation of MBCP/fibrin glue composites. These bone substitutes exhibiting osteoinductive properties could be used for the reconstruction of large bone defects.     

可注射磷酸钙骨水泥及其应用

可注射磷酸钙骨水泥作为一种新型人工骨替代材料,以其良好的生物相容性和骨传导性被广泛应用于临床骨缺损和牙缺损的修复.本文介绍了可注射磷酸钙骨水泥的种类和特性,指出了存在的问题和应用前景.     

Biomimetic properties of an injectable chitosan/nano-hydroxyapatite/collagen composite

To meet the challenges of designing an injectable scaffold and regenerating bone with complex three-dimensional (3D) structures, a biomimetic and injectable hydrogel scaffold based on nano-hydroxyapatite (HA), collagen (Col) and chitosan (Chi) is synthesized. The chitosan/nano-hydroxyapatite/collagen (Chi/HA/Col) solution rapidly forms a stable gel at body temperature. It shows some features of natural bone both in main composition and microstructure. The Chi/HA/Col system can be expected as a candidate for workable systemic minimally invasive scaffolds with surface properties similar to physiological bone based on scanning electron microscopic (SEM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR) results.     

Influence of calcium chloride and aprotinin in the in vivo biological performance of a composite combining biphasic calcium phosphate granules and fibrin sealant

Highly bioactive biomaterials have been developed to replace bone grafts in orthopedic revision and maxillofacial surgery for bone augmentation. A mouldable, self-hardening material can be obtained by combining TricOs Biphasic Calcium Phosphate Granules and Tissucol Fibrin Sealant. Two components, calcium chloride and antifibrinolytic agents (aprotinin), are essential for the stability of the fibrin clot. The ingrowth of cells in composites combining sealants without calcium chloride or with a low concentration of aprotinin was evaluated in vivo in an experiment on rabbits. Bone colonization was compared using TricOs alone or with the composite made from TricOs and the standard fibrin sealant. Without the addition of calcium chloride, the calcium ions released by the ceramic component interacted with the components of the sealant too late to stabilize the clot. With a low concentration of aprotinin, the degradation of the clot occurred more quickly, leading to the absence of a scaffold on which the bone cells could colonize the composite. Our results indicate that a stable fibrin scaffold is crucial for bone colonization. The low calcium chloride and low aprotinin groups have shown lower bone growth. Further studies will be necessary to determine the minimal amount of antifibrinolytic agent (aprotinin) necessary to allow the same level of osteogenic activity as the TricOs-fibrin glue composite.     

Current state of the art of biphasic calcium phosphate bioceramics

We have developed 15 years ago, with the collaboration of Lynch, Nery, and LeGeros in the USA, a bioactive concept based on biphasic calcium phosphate (BCP) ceramics. The concept is determined by an optimum balance of the more stable phase of HA and more soluble TCP. The material is soluble and gradually dissolves in the body, seeding new bone formation as it releases calcium and phosphate ions into the biological medium. The bioactive concept based on the dissolution/transformation processes of HA and TCP has been applied to both Bulk, Coating and Injectable Biomaterials. The events at the calcium phosphate (CaP) biomaterial/bone interface represent a dynamic process, including physico-chemical processes, crystal/proteins interactions, cells and tissue colonization, bone remodeling, finally contributing to the unique strength of such interfaces. An important literature and numerous techniques have been used for the evaluation of the fundamental physico chemical and biological performance of BCP concept. This type of artificial bone used from a long time in preclinical and in clinical trial, revealed the efficiency for bone filling, performance for bone reconstruction and efficacy for bone ingrowth at the expense of the micro macroporous BCP bioceramics.     

Laser engineered multilayer coating of biphasic calcium phosphate/titanium nanocomposite on metal substrates

In this work, laser coating of biphasic calcium phosphate/titanium (BCP/Ti) nanocomposite on Ti-6Al-4 V substrates was developed. A continuous wave neodymium-doped yttrium aluminium garnet (Nd:YAG) laser was used to form a robust multilayer of BCP/Ti nanocomposite starting from hydroxyapatite and titanium nanoparticles. In this process, low power coating is realized because of the strong laser-nanoparticle interaction and good sinterability of nanosized titanium. To guide the optimization of laser processing conditions for the coating process, a multiphysics model coupling electromagnetic module with heat transfer module was developed. This model was validated by laser coating experiments. Important features of the coated samples, including microstructures, chemical compositions, and interfacial bonding strength, were characterized. We found that a multilayer of BCP, consisting of 72% hydroxyapatite (HA) and 28% beta-tricalcium phosphate (β-TCP), and titanium nanocomposite was formed on Ti-6Al-4 V substrates. Significantly, the coating/substrate interfacial bonding strength was found to be two times higher than that of the commercial plasma sprayed coatings. Preliminary cell culture studies showed that the resultant BCP/Ti nanocomposite coating supported the adhesion and proliferation of osteoblast-like UMR-106 cells.     

海藻酸钙水凝胶/聚乳酸复合材料的制备与性能

通过化学发泡-冷冻干燥-粒子滤出复合法制备聚乳酸(PLLA)大孔支架, 然后在大孔内以海藻酸钠(SA)、碳酸钙、葡萄糖酸内酯(GDL)为原料, 通过原位相转变制备海藻酸钙水凝胶/聚乳酸复合材料(CA/PLLA); 分别利用SEM、压缩强度测试和细胞培养对CA/PLLA支架的形貌、力学性能及生物相容性进行了研究。结果表明: PLLA具有直径小于2 mm、孔道相互连通的孔洞, 且在大孔中能够形成均匀的CA。CA/PLLA复合材料的压缩强度(2.74 MPa)远大于单一的海藻酸钙水凝胶的压缩强度(0.10 MPa)。在CA/PLLA复合支架中, 软骨细胞呈簇状圆形生长状态, 与其在天然软骨陷窝里生长状态一致。这种软硬结合、天然与合成高分子杂化的CA/PLLA复合材料的力学强度和生物相容性同时得到提高, 可进一步作为骨和软骨修复材料研究。     

Macroporous biphasic calcium phosphate ceramics versus injectable bone substitute: a comparative study 3 and 8 weeks after implantation in rabbit bone

Macroporous biphasic calcium phosphate ceramics (MBCP) and a calcium phosphate injectable bone substitute (IBS), obtained by the association of biphasic calcium phosphate (BCP) ceramic granules and an aqueous solution of a cellulosic polymer, were compared in the same animal model. The two tested biomaterials were implanted in distal femoral osseous defects in rabbits. Qualitative and quantitative histological evaluation was performed three and eight weeks after implantation to investigate bone colonization and ceramic biodegradation associated with the two bone substitutes.Both biomaterials expressed osteoconduction properties and supported the apposition of a well-mineralized lamellar newly-formed bone. Bone colonization occurred much earlier and faster for IBS than for MBCP implants, although the respective rates of newly-formed bone after eight weeks of implantation did not differ significantly. For both biomaterials, ceramic resorption occurred regularly throughout the implantation period, though to a greater extent with IBS than with MBCP implants.The associated polymer in IBS produced intergranular spaces allowing body fluids to reach each BCP ceramic granule immediately after implantation, which may have favored osteoblastic activity, new bone formation and ceramic resorption. This completely interconnected open macroporosity could account for the earlier and more satisfactory bone substitution achieved with IBS. © 2001 Kluwer Academic Publishers     

不同孔隙率的双相磷酸钙陶瓷降解性能研究

包含羟基磷灰石(HA)和磷酸三钙(TCP)的双相磷酸钙陶瓷(BCP)由于其具有良好的降解性能和良好的骨诱导性被看作是骨替代和修复的首选材料。BCP陶瓷的降解性能主要受相成份、孔隙率、材料微观形貌的影响。通过化学共沉淀法制得了成份为60/40(HA/TCP)的双相BCP粉体,通过双氧水发泡法制得了孔隙率分别为40%、60%和80%的多孔双相BCP陶瓷。选用Tris缓冲液浸泡的方法测试材料的体外降解行为。结果显示,孔隙率的改变有效地调控了BCP陶瓷的降解性能。随孔隙率的增加材料的溶出显著加快。高孔隙率材料的快速降解,在体系中释放出相对较高的钙、磷浓度,这可能是其高生物活性的重要影响因素。     

Production of in-situ macropores in an injectable calcium phosphate cement by introduction of cetyltrimethyl ammonium bromide

In the present study, cetyltrimethyl ammonium bromide (CTAB) was introduced to an injectable calcium phosphate cement (CPC) to produce macropores during the setting process to accelerate the absorbing ability in vivo. The effects of CTAB on the rheological properties, injectability, setting time, compressive strength, phase evolution, microstructure and degradation rate of CPC were studied. The results showed that the addition of CTAB increased the viscosity and yield stress, and decreased the injectability of the cement pastes. The macroporosity and total porosity increased and the compressive strength of the cement obviously decreased with the increase of CTAB. The macroporosity of the CPC prepared at 5 mM CTAB solution reached 44.2 +/- 2.5% and the mass loss of the cement increased almost 50% as compared with the cement without CTAB. Considering the injectability, compressive strength and degradation rate of CPC, the preferred CTAB concentration was 5 mM. The injectable CPC with macropores is promising to be used in minimally invasive approach.     

Hyaluronic acid/mildly crosslinked alginate hydrogel as an injectable tissue adhesion barrier

Although hyaluronic acid (HA) has been conventionally utilized as a tissue adhesion barrier material, its rapid clearance in the body still remains as a big challenge in the clinical practice. In this study, we prepared a hydrogel of HA embedded in mildly crosslinked alginate (HA/mcALG hydrogel), which is injectable, easily covers injured tissues, and remains stably at the applied site during wound healing (by muco-adhesive HA embedded in the network structure of the mcALG hydrogel). The HA/mcALG hydrogel was highly effective for the prevention of peritoneal tissue adhesion compared to HA and mcALG hydrogels, and did not lead to any abnormal tissue responses during wound healing. The HA/mcALG hydrogel can be a good candidate as an injectable tissue adhesion barrier for clinical applications.