| Introduction of Background Fractures, bone tumors, the aged-related osteoporosis and revision of arthroplasty are still considerd to be the most common and frequently encountered diseases in the field of orthopedics. In the treatment of these diseases , bonegraft have often been used because of bone defect, which will increase with an aging population.Next to blood, bone is the second most transplanted tissue. Autograft is currently regarded as the "gold standard" because it possesses all three of the essential elements,osteoconductive matrix, osteogenic stem cells and osteoinductive factors,which are needed for an optimal bone graft . The use of autograft has drawbacks, as it requires a second surgical exposure to collect the bone tissue. This involves increased operative time, additional cost, postoperative infection,pain, haemorrhage and nerve injury . Only a limited amount of autograft bone tissue is available . Because of this, allograft and xenografts are widely used clinically . Both of allografts and xenografts not only bear immunologic rejection ,but also a risk of transmisson of infections.With all the problems above,:more and more interests focused on the synthetic bone substitute. Apart from sufficient supply, no bone bank needed, synthetic bone substitutes could have a controlled quality and be designed to have optimised mechanical and biological properties for specific applications. They could also be shaped to fit the defect, or even better, be used with minimally invasive surgical techniques, hardening in situ and giving stability for any defect geometry.At present,the most common used synthetic bone substitutes included calcium sulphate(CaS) and calcium phosphate based bone substitutes.It have been a long time for calcium sulphate to be used as a bone defect filling. Calcium sulphate showed good biocompatibility, but was resorbed quickly and had low strength,which making it less useful in larger or non-contained defects. Calcium phosphates have been studied as bone repair materials for more than 100 years. In 1983, Brown and Chow discovered the first calcium phosphate cement(CPC) and placed on the use of it for bone substitute. With the different composition, CPCs can be used for time-controlled biodegradation in vivo. Because they possess a very large specific surface area, CPC will biodegrade much more active than traditional CaP. Unfortunately, the properies of currently available CaP cements are still insufficient for reliable and safe clinical application. Problems have been reported with the setting time, mechanical strength, application technique, lossing of cohesion after contact with body fluids, and final biological properties.Calcium phosphate showed a slow resorption,but calcium sulphate were resorbed too rapidly, both of which were not compatible with new bone formation.It could be reasonable to use a compound of calcium phosphate with calcium sulphate as a filler for bone defect. Injectable CaP with CaS bone substitute has been considered as a porous-crated injectable material. The combination of CaP and CaS regulates material resorption rate, allowing rapid bone regeneration.Objectives To investigate the physicochemical properties, bone regeration and integration characteristics of a-tricalcium phosphate and a-tricalcium phosphate with 20% calcium phosphate in bone harvest chambers and trabecular bones.To evaluate the biomechanical characteristics of both materials using a indentation test. To investigate bone integration and material degradable characteristics of a-TCP with 1% Si in bone harvest chamber.Materials and Mathods1.Preparation of injectabe materialsa-TCP(injectable bone substitute-1,IBS-1) was produced by mixing calcium phosphate and calcium carbonate carbonate at a 2:1 molar ratio. The two powders were mixed together mechanically for 15 min,heated up to 1300℃for 12 h, and then quenched in air. The material obtained was ground to powder in a ball mill and checked for purity by X-ray diffraction (XRD). The obtained median particle size(D0.5) was 7μm.α-TCP is mixed with 20-wt%α-calcium sulphate hemihydrate or with 1% Silicon(Si). The materials were sterilized and prepared by mixing the powder with 2.5% Na2HPO4 at a L/P ratio of 0.34 ml g-1 for IBS-1 or a-TCP-Si, and 0.36 ml g-1 for a-TCP with 20%CaS(injectable bone substitute-2,IBS-2).2.Measuring the setting time of IBS-1 and IBS-2The initial setting time and final setting time of both material were measured using Gillmore Needles instrument.The measurements were divided into three groups according to the condition.Two measurements were under the condition of 21℃with 50% or 100% humidity and the other one was 37℃with 100% humidity.Each group incliudes nine samples.3. Compressive TestAfter being stored in Teflon-moulds for 1 week,the samples with 4mm in diameter and 8mm in height, were taken out and tested in compression using an Universal Testing Machine at a speed of 1 mm.min-1 until failure.4. After the compressive test finished,the pieces of the materials was hand crushed using a pestle and mortar to a fine powder before analysis. XRD patterns of the samples were recorded.The data were analysed with diffraction standards to identify the chemical composition, 5. Bone integration and regeneration in bone harvest chamber(BHC)The BHC is a titanium cylinder,7mm in height and 6mm in diameter, which bear a screw thread on outside wall and contains a core with a 1×1×5 mm groove facing the bottom of the cylinder.This groove is coaxial with holes in the outer cylinder, providing a continuous canal through the entire device for tissue ingrowth. When the core is removed, the tissue inside the bone ingrowth canal is uncovered and can be harvested without disturbing the surrounding bone. The chamber can be used for repeated harvesting. Both IBS-1 and IBS-2 were separatedly implanted in pairs, one sample and one empty control, in the BHCs in each rabbit.Tissue was harvested at 3 and 6 weeks. The harvested tissue was decalcified and stained with hematoxylin and eosin,or Trap. The type, amount and location of bone and fibrous tissue were noted.The amounts of new bone formation and material degradation were measured by an imaging system.6. Histological study on the injectable a-TCP with 1% siliconBone harvest chambers were implanted bilaterally in the proximal tibial of six mature rabbits, a-TCP+1wt%silicon was implanted in the experimental group, and only a-TCP was implanted in the control group. The tissue,harvested from the chambers at 1,2,3 weeks after implantation,were evaluated by light microscopy and histomorphometry. The number of osteoclasts, bone formation and the new bone integration with the materials were analyzed in histological specimens with HE, Goldner and TRAP staining.7. Forty five male Sprague-Dawley rats were usedHoles of 3 mm diameter and 4 mm deep were drilled by a hole-cutter in both bilateral tibiae and femora. The materials were randomly injected into the bone defects. The holes were covered by a silicon cap fixed by a "u" pin. For each time point (1,4,7,21,42 and 84 days), six rats were given the materials, and a sham operation was performed on one animal, by drilling and leaving the hole empty. In order to measure the strength of cancellous bone, the cortex on the tibia was removed by the same hole-cutter in a further 6 tibiae.Histological study The implanted and control femora were prepared for histology 7 days and after. The specimens were decalcified and H&E staining. New bone formation and material remaining in the bone defect were identified under scanning electronic microscope.Indentation test The resected tibiae were placed on an Instron 8511. A 2 mm diameter metal rod was positioned above the centre of the hole and moved down at a speed of 1 mm min-1. The indentation load was recorded. The mean of each point in all 6 samples was calculated. The same tests were also applied to empty control group and cancellous bone.Results It was confirmed by XRD that the injectable bone substitutes tested were IBS-1 and IBS-2.The results of Gillmore Needles testing showed that temperature and humidity could change the initial as well as final setting time of the both biomaterials.The lower temperature,the longer setting time;the more humidity, the longer setting time.Under the same temperature and humidity, IBS-2 had shorter setting time than that of IBS-1.Under the condition of 37℃and 100% in humidity, the initial and final setting time of IBS-1 went to 34 and 53 minutes.Under the same condition,that of IBS-2went to 22 and 44 minutes.The compressive sthength of IBS-1 was 33.8(range from 23.5 to 43.2) Mpa and of IBS-2 was 22/5(range from 20.2 to 26.5) Mpa.In the chambers containingIBS-1 andIBS-2, new trabecular bone was formed inside the chamber. No inflammatory reaction or fibrous tissue was observed between the material and the new bone. Some osteoblasts and osteoclasts were observed in the new bone attached to the materials. No significant difference was found between 3 and 6 weeks with different materials in the proportion of bone formation and residual material.The material with a-TCP+1%silicon contacts tightly with the surrounding bone tissues.No inflammation and fibrous tissue was observed during observation period. The number of osteoclasts, the bioactivity of osteoblasts and osteointegration around the materials were increased in the tissue with a-TCP+1wt% silicon compared with a-TCP.In reparing the bone defects of rats histology On day 7, the bone defect without any material was only filled by blood. With materials, blood cells penetrated partly into IBS-1, most of which were separated into pieces,but not in IBS-2. By day 21, new bone integrated into both materials. And by day 42, newly formed bone was mature and formed a thin layer of compact bone in the defect opening. By day 84, the compact bone had become thicker compared with day 42. The remaining materials were surrounded by newly formed bone that occupied the most of the bone defect area. Compared to IBS-1, less IBS-2 remained by day 84.Indentation test Compared to cancellous bone, the bone defect area without filling showed no strength at days 1, 4 and 7; after day 21, a force above 20 N was generated after a small displacement which then decreased rapidly with further displacement. In the bone defect with the materials In days 1 and 4, forces from IBS-1 and IBS-2 were the same as cancellous bone. By day 7 and the period after, the forces of the two materials were much higher compared with cancellous bone. A statistic analysis showed that local loading was significantly increased after day 7 compared to cancellous bone.Conclusions The IBS-1 and IBS-2 in these experiments could be used as bone substitutes because of the characteristics of good injectability, biocompadibility, osteoconduction, bone regeneration, biodegradability and absorbability. After 7 days,the compression strength of both materials have stronger than that of cancellus bone of rats.Compared with IBS-1,IBS-2 owns the characteristics of shorter setting time,more bone formation and faster biodegradability.The a-TCP with 1% silicon also showed more bone regenerated and biodegradable than IBS-1 did.
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