Engineered synovial joint condyle using demineralized bone matrix

In an effort to develop tissue-engineered bio-joints, a novel demineralized joint scaffold was achieved by peeling off the cartilage layer of a distal femur joint condyle. Primary chondrocytes were then seeded onto the demineralized joint condyle scaffolds and cultured in vitro for 6 weeks. Histological staining and biochemical assays of the engineered joints showed that after 6 weeks in vitro culture, a cartilaginous layer had formed on the demineralized joint scaffold that was similar to native synovial articular cartilage with respect to palpation and texture. Meanwhile, the engineered joint condyle cartilage demonstrated rudimentary morphological and structural resemblance to native cartilage. Intense and uniform safranin-O red staining was found in engineered joint condyle cartilage. Furthermore, glycosaminoglycan (GAG) assays confirmed that there were no statistical differences in the GAG/DNA ratio between the engineered joint cartilage and native cartilage (p > 0.05). In conclusion, a novel scaffold and a practical method have therefore been developed for total joint tissue engineering based on demineralized bone scaffold. The morphological appearance of the engineered joint and the rudimentary biochemical quantification resemble that of a native articular condyle.     

Fluorapatite-coated implants in experimental arthritis: the response of rabbit trabecular bone

Fluorapatite-coated implants have been studied for the first time under non-optimal tissue conditions and have shown promising results. The influence of arthritis on the tissue response to implants coated with fluorapatite (FA) was studied in an arthritis model. Immune complex-induced arthritis was elicited in the right knee-joint of eight rabbits while the contralateral joint served as control. Ti-6Al-4V cylinders, plasma-spray coated with FA were implanted in the patellar groove (PG) and medial femoral condyle (MC) in each knee for 6 weeks. Histology showed a close bone-to-implant contact at the lateral surface of the implants without any intervening soft tissue or inflammatory cells. Histomorphometry revealed no differences in bone apposition between control and arthritic joints, but the MC-implants showed more bone apposition than the PG-implants. Parts of the implant surface were not covered by bone, but were in contact with bone marrow. The FA coating on the implant sides did not show signs of resorption in the control and arthritic joints, but the coating on the upper surface of the implants was partially resorbed in both the control and arthritic joints. The arrangement and composition of the regenerating tissue in this location was profoundly influenced by the inflammatory process in the arthritic joints. In a previous study, using the same arthritis model, an impaired bone formation was found around commercially pure titanium implants in arthritic joints. In the present study, the unimpaired bone formation around FA-implants in the arthritic joints indicates that an FA coating adds advantageous properties to metal implants used in tissues influenced by an on-going inflammation.     

Evaluation of a press-fit osteochondral poly(ester-urethane) scaffold in a rabbit defect model

The purpose of this study was to evaluate the impact on osteochondral healing of press-fitted multiphasic osteochondral scaffolds consisting of poly(ester-urethane) (PUR) and hydroxyapatite into a cylindric osteochondral defect in the distal non-weight bearing femoral trochlear ridge of the rabbit. Two scaffolds were investigated, one with and one without an intermediate microporous membrane between the cartilage and the bone compartment of the scaffold. A control group without a scaffold placed into the defect was included. After 12 weeks macroscopic and histomorphological analyses were performed. The scaffold was easily press-fitted and provided a stable matrix for tissue repair. The membrane did not demonstrate a detrimental effect on tissue healing compared with the scaffold without membrane. However, the control group had statistically superior healing as reflected by histological differences in the cartilage and subchondral bone compartment between control group and each scaffold group. A more detailed analysis revealed that the difference was localized in the bone compartment healing. The present study demonstrates that an elastomeric PUR scaffold can easily be press-fitted into an osteochondral defect and provides a stable matrix for tissue repair. However, the multi-phasic scaffold did not provide a clear advantage for tissue healing. Future investigations should refine especially the bone phase of the implant to increase its stiffness, biocompatibility and osteoconductive activity. A more precise fabrication technique would be necessary for the matching of tissue organisation.     

Optimum design of artificial joints considering initial fixation of prosthesis

Living bone is a stratified structural system which is functionally built of many substructures. Loads applied on the articular surface are transmitted through the articular cartilage, the subchondral bone and the cancellous bone to the shaft bone. The prosthesis for the artificial joint is a material replacing the articular cartilage and the subchondral bone. So, it is important for an effective design of the artificial joint possessing the mechano-compatibility to living bones to understand the mechanical behavior of them sufficiently. The purpose of this paper is to investigate the optimum design of the artificial joint by considering the better plate shapes of the prosthesis for the fixation on the cancellous bone under compressive loading based on the mechanical behavior of the subchondral bone. Consequently, the effectiveness of the optimum designs of the artificial joints using the arched plate prostheses is shown numerically.     

Structure of the interface between rabbit cortical bone and implants of gold, zirconium and titanium

The role of surface properties (chemical and structural) for the interaction between biomaterials and tissue is not yet understood. In the present study, implants made of titanium, zirconium (transition metals with surface oxides) and gold (metallic surface) were inserted into the rabbit tibia. Light microscopic (LM) morphometry showed that after 1 and 6 mo the gold implants had less amount of bone within the threads and a lower degree of bone-implant contact than the titanium and zirconium implants, which did not differ from each other. These quantitative differences were supported by LM and ultrastructural observations of the interface. The ultrastructural observations in addition demonstrated that the layer of non-collagenous amorphous material located between the implant and the calcified bone was appreciably thicker around zirconium than around titanium implants. The factors potentially responsible for the observed morphological differences in the bone around the different material surfaces are discussed.     

The regeneration of articular cartilage using a new polymer system

A polymer system based on room temperature polymerising poly (ethylmethacrylate) polymer powder and tetrahydrofurfuryl monomer has been investigated as a biomaterial for encouraging articular cartilage repair. This heterocyclic methacrylate polymer system swells slightly in situ and thus provides a good interface with subchondral bone resulting in mechanical stability with favourable uptake kinetics. Another feature of this polymer system is that it exhibits high water uptake which leads to absorption of the surrounding tissue fluid and matrix proteins, including growth factors; this may encourage the formation of new cartilage. Three weeks after implantation the tissue overgrowth contained cartilage components: chondrocytes, collagen type II, chondroitin 4-sulphate and chondroitin 6-sulphate. In addition numerous chondrocyte clones were observed at the edge of the defect and in the newly repaired tissue. By six weeks a superficial articulating surface was continuous with the normal articular cartilage with underlying tissue which showed some evidence of endochondral ossification. By nine weeks the surface covering of new cartilage had a widened and an irregular zone of calcified cartilage with thickened subchondral bone was present. At eight months the resurfaced cartilage remained intact above a remodelled subchondral bone end plate.     

An evaluation of chondrocyte morphology and gene expression on superhydrophilic vertically-aligned multi-walled carbon nanotube films

Cartilage serves as a low-friction and wear-resistant articulating surface in diarthrodial joints and is also important during early stages of bone remodeling. Recently, regenerative cartilage research has focused on combinations of cells paired with scaffolds. Superhydrophilic vertically aligned carbon nanotubes (VACNTs) are of particular interest in regenerative medicine. The aim of this study is to evaluate cell expansion of human articular chondrocytes on superhydrophilic VACNTs, as well as their morphology and gene expression. VACNT films were produced using a microwave plasma chamber on Ti substrates and submitted to an O₂ plasma treatment to make them superhydrophilic. Human chondrocytes were cultivated on superhydrophilic VACNTs up to five days. Quantitative RT-PCR was performed to measure type I and type II Collagen, Sox9, and Aggrecan mRNA expression levels. The morphology was analyzed by scanning electron microscopy (SEM) and confocal microscopy. SEM images demonstrated that superhydrophilic VACNTs permit cell growth and adhesion of human chondrocytes. The chondrocytes had an elongated morphology with some prolongations. Chondrocytes cultivated on superhydrophilic VACNTs maintain the level expression of Aggrecan, Sox9, and Collagen II determined by qPCR. This study was the first to indicate that superhydrophilic VACNTs may be used as an efficient scaffold for cartilage or bone repair.     

Site-Dependent Osseointegration of Biodegradable High-Purity Magnesium for Orthopedic Implants in Femoral Shaft and Femoral Condyle of New Zealand Rabbits

Magnesium(Mg) has been widely accepted as osteoconductive biomaterial, but osseointegration of Mg device at different implantation sites is still unclear. In the present study, high-purity magnesium(HP Mg)pins were implanted into femoral shaft and condyle of New Zealand rabbits concurrently. 2, 8, 12 and 16 weeks after surgery, rabbit femurs were harvested for micro-computed tomography(micro-CT) scanning and subsequent histological examinations. HP Mg pins were retrieved for scanning electron microscope and energy dispersive spectrum(SEM/EDS) analyses. HP Mg pins at both implantation sites performed stable corrosion with mineral deposition and bone incorporation on surface. However, difference in distribution of contact osteogenesis centers and biological properties of peri-implant bone tissues was detected between femoral shaft and femoral condyle. In femoral condyle, contact osteogenesis centers originated from both periosteum and cancellous bones and the whole HP Mg pin was encapsuled in trabecular bone at 16 weeks.Meanwhile, bone volume to total bone volume(BV/TV) and bone mineral density(BMD) of peri-implant bone tissues were above those of normal bone tissues. In femoral shaft, contact osteogenesis centers were only from periosteum and direct bone contact was confined in cortical bone, while BV/TV and BMD kept lower than normal. Furthermore, new formation of peri-implant bone tissues was more active in femoral condyle than in femoral shaft at 16 weeks. Therefore, although HP Mg performed good biocompatibility and corrosion behavior in vivo, its bioadaption of osseointegration at different implantations sites should be taken into consideration. Bone metaphysic was suitable for Mg devices where peri-implant bone tissues regenerated rapidly and the biological properties were close to normal bone tissues.     

A hydrophylic polymer system enhanced articular cartilage regeneration in vivo

This study describes a new method for the repair of large articular cartilage defects in the knee joint and compares the effect of two polymer systems on the quality of the repair tissue. The two systems are a newly developed hydrophylic system, based on poly-ethyl-methacrylate (PEMA) polymer and tetra-hydro-furfuryl-methacrylate (THFMA) monomer and the conventional bone cement polymer system, based on poly-methyl-methacrylate (PMMA) polymer and methyl-methacrylate (MMA) monomer. Thirty adult Sandy-lop rabbits were used. Both knees were operated on in each animal, the one defect received either PEMA/THFMA or conventional bone cement and the contralateral defect received no biomaterial (control group). Femora were retrieved at six weeks and the repair tissue was studied by histology, histochemistry and immuno-histochemistry. PEMA/THFMA enhanced the quality of the repair significantly (p<0.0001). By six weeks hyaline-like articular cartilage was the predominant tissue covering the defects and it was fully integrated with the surrounding normal articular cartilage. Immuno-localization showed cartilage components, including collagen type II, distributed evenly throughout its matrix. PMMA/MMA on the other hand did not improve significantly the repair tissue, which was predominately fibro-cartilaginous, poorly bonded to the adjacent normal articular cartilage. The method of implantation is simple and easily reproducible and the new polymer has been well-accepted by the rabbits.     

Studies of the healing of bone grafts, and the incorporation of titanium implants in grafted bone: an experimental animal model

An insufficient quality and amount of bone often necessitate the clinical use of implants together with bone transplants. The present study describes an experimental animal model for the study of implants in bone grafts. Circular defects were made bilaterally in the tibia of 36 rabbits. The defects received either autologous cortical bone (control), demineralized bone matrix (DBM), plasma-augmented DBM or were left empty (without bone graft). In all defects a titanium implant was centrally placed and anchored in the opposite cortex. Evaluation with light microscopic morphometry showed that the insertion of a threaded titanium implant centrally in a cortical defect was followed by a spontaneous healing of the defect after 6 mon. After 6 wk, all implants in cortical grafts were well integrated with a significantly higher bone-to-implant contact than in the DBM and plasma-augmented groups. After 6 mon, all experimental groups had a mean bone area within the threads ranging between 69% and 80% and a mean bone-to-implant contact between 31% and 42%. The results from the present study indicate that the model allows comparative studies on the early formation, resorption and remodelling of bone around implants after modification of implant, graft and host properties.     

实时超声膨胀测量系统在关节软骨中的实验研究

关节软骨是覆盖关节表面的一层承载生物重量的组织。关节软骨在正常状态下，软骨蛋白多糖的弹性和胶原纤维的张力保持平衡。这种平衡的微小变化会引起关节软骨的退化。关节软骨特别是其表层区域的膨胀是和骨关节炎的退化相联系的，定量的测量关节软骨的膨胀可以表征骨关节炎的退化变化。本文的主要目的是介绍一种新的实时超声膨胀测量系统，并把该系统应用到关节软骨的研究中。该系统使用50MHz的超声来实时动态观测用胰岛素和0．15M生理盐水溶液分别浸泡处理的牛膝盖关节软骨的非均匀瞬时深度依赖膨胀行为。实验表明，实时超声检测的方法为关节软骨的退化研究提供了独特的工具。这项技术结合关节内窥镜检查，具有潜在的早期诊断体内关节退化的价值。     

Repair of large osteochondral defects in a beagle model with a novel type I collagen/glycosaminoglycan-porous titanium biphasic scaffold

The limited repair potential of articular cartilage, which hardly heals after injury or debilitating osteoarthritis, is a clinical challenge. The aim of this work was to develop a novel type I collagen (Col)/glycosaminoglycan (GAGs)-porous titanium biphasic scaffold (CGT) and verify its ability to repair osteochondral defects in an animal model with bone marrow stem cells (bMSCs) in the chondral phase. The biphasic scaffold was composed of Col/GAGs as chondral phasic and porous titanium as subchondral phasic. Twenty-four full-thickness defects through the articular cartilage and into the subchondral bone were prepared by drilling into the surface of the femoral patellar groove. Animals were assigned to one of the three groups: 1) CGT with bMSCs (CGTM), 2) only CGT, and 3) no implantation (control). The defect areas were examined grossly, histologically and by micro-CT. The most satisfied cartilage repairing result was in the CGTM group, while CGT alone was better than the control group. Abundant subchondral bone formation was observed in the CGTM and CGT groups but not the control group. Our findings demonstrate that a composite based on a novel biphasic scaffold combined with bMSCs shows a high potential to repair large osteochondral defects in a canine model.     

An evaluation of human articular cartilage on femoral head

In this study, we investigated stress relaxation behavior of the human articular cartilage on femoral head. Articular cartilage is a white dense connective tissue that covers the bone ends within diarthrodial joints and works as a weight-transmitting and energy-absorbing material. Human articular cartilage on femoral head was used as test material. Relaxation tests were carried out by using the indentation technique via Instron Universal Testing Machine. Test materials were investigated in an isotonic salt solution at 37 °C. To keep the temperature constant, two vessels being in each other were utilized. Thus, hot water was circulated in the outer vessel and isotonic salt solution was kept in the inner vessel. Experimental results showed that there is a remarkable difference between normal and degenerated cartilage for the same age and sex. It was observed that the relaxation percent of normal cartilage as a function of relaxation time is much higher than that of degenerated cartilage.     

Characterization of the repair tissue in articular cartilage defects using silver-enhanced colloidal gold immunostaining

The newly developed silver-enhanced colloidal gold staining method was used in a rabbit model to characterize the repair tissue in large articular cartilage defects filled with a heterocyclic methacrylate polymer. By 6 weeks the resurfacing tissue consisted of highly organized hyaline-like articular cartilage, fully integrated with the adjacent normal cartilage. Immuno-histochemistry detected collagen type ll, keratan sulphate, chondroitin 4-sulphate and chondroitin 6-sulphate in the matrix of the neocartilage. The level to which the polymer plug was recessed apeared to be critical to the overall quality of the repair tissue. Optimum results were obtained when the top surface of the biomaterial was at the level of the subchondral bone, below the level of the surrounding articular cartilage. Other technical aspects of implantation, that also affect the repair, are also discussed.     

Zone-dependent mechanical properties of human articular cartilage obtained by indentation measurements

Emerging 3D printing technology permits innovative approaches to manufacture cartilage scaffolds associated with layer-by-layer mechanical property adaptation. However, information about gradients of mechanical properties in human articular cartilage is limited. In this study, we quantified a zone-dependent change of local elastic modulus of human femoral condyle cartilage by using an instrumented indentation technique. From the cartilage superficial zone towards the calcified layer, a gradient of elastic modulus values between 0.020?±?0.003?MPa and 6.44?±?1.02?MPa was measured. To validate the tissue quality, the histological tissue composition was visualized by glycosaminoglycan and collagen staining. This work aims to introduce a new protocol to investigate the zone-dependent mechanical properties of graded structures, such as human articular cartilage. From this knowledge, better cartilage repair strategies could be tailored in the future.     

Healing of titanium implants in onlay bone grafts: an experimental rabbit model

An experimental rabbit bone graft model for the study of bone formation and remodeling around titanium implants is described. A 2.5-cm long radius bone segment served as an onlay graft. Two commercially pure (c.p.) titanium implants were inserted into the bone graft prior to fixation to the inferior border of the mandibular base with osteosynthesis titanium screws. Each animal was operated twice, allowing follow-up periods of 6 weeks on one side and 6 months on the contralateral side. In order to study bone remodeling by means of fluoroscopy the animals received single injections of tetracyclin and alizarine complexone 2 weeks and 1 week, respectively, prior to sacrifice by perfusion fixation with glutaraldehyde. The bone and implants were excized en bloc, postfixed and embedded in plastic resin. Stained and unstained thin ground sections as well as microradiographed thick sections were produced for light microscopic morphometry and fluoroscopy. After 6 weeks, osteoclastic/osteoblastic activity was primarily observed in the graft-recipient contact area and in the intracortical compartment of the graft bone. New bone formation observed on the implant surface originated from the recipient site. The bone formation was evident also in the implant-graft interface. At 6 weeks the average bone fill of the implant threads was 28.4% which increased to 36.4% after 6 months as measured by morphometry. An average of 17.6% bony contact was measured after 6 weeks which increased to 29.7% 6 months after surgery. The graft bone had reduced in size from an average of 39.5% after 6 weeks down to 24.8% after 6 months (P < 0.05). It is concluded that the described experimental model can serve as a useful method for the study of implant healing in onlay grafts.     

Preparation of a biphasic scaffold for osteochondral tissue engineering

Tissue engineering has been developed as a prospective approach for the repair of articular cartilage defects. Engineered osteochondral implants can facilitate the fixation and integration with host tissue, and therefore promote the regeneration of osteochondral defects. A biphasic scaffold with a stratified two-layer structure for osteochondral tissue engineering was developed from biodegradable synthetic and naturally derived polymers. The upper layer of the scaffold for cartilage engineering was collagen sponge; the lower layer for bone engineering was a composite sponge of poly(DL-lactic-co-glycolic acid) (PLGA) and naturally derived collagen. The PLGA–collagen composite sponge layer had a composite structure with collagen microsponge formed in the pores of a skeleton PLGA sponge. The collagen sponge in the two respective layers was connected. Observation of the collagen/PLGA–collagen biphasic scaffold by scanning electron microscopy (SEM) demonstrated the connected stratified structure. The biphasic scaffold was used for culture of canine bone-marrow-derived mesenchymal stem cells. The cell/scaffold construct was implanted in an osteochondral defect in the knee of a one-year old beagle. Osteochondral tissue was regenerated four months after implantation. Cartilage- and bone-like tissues were formed in the respective layers. The collagen/PLGA–collagen biphasic scaffold will be useful for osteochondral tissue engineering.     

N-乙酰氨基葡萄糖促进骨缺损愈合作用的研究

探讨了甲壳素降解产物N—乙酰氨基葡萄糖对骨缺损愈合的促进作用。将N—乙酰氨基葡萄糖以一定浓度加入培养基中，观察其对体外培养的成骨细胞株MC3T3—E1的促进生长增殖的作用。结果发现，N—乙酰氨基葡萄糖在适当浓度均能促进体外培养的成骨细胞增殖，其中1000mg／L时作用尤为明显。手术造成新西兰兔右侧桡骨3mm缺损，随机分成对照组、阳性药物组和N—乙酰氨基葡萄糖卖验组，每日灌胃给药。分别于术后第9天、17天、30天、42天取材，通过各时段X线摄片结果和组织学切片可以观察到，买验组的血肿机化、吸收，软骨性骨痂、软骨内化骨和小梁骨形成及骨损伤愈合方面明显早于对照组。结果提示N—乙酰氨基葡萄糖对促进骨缺损的愈合有显著的作用，有望成为临床治疗骨缺损的有效药物。     

Hydroxyapatite-ceramic for juxta-articular implantation

The histocompatibility of hydroxyapatite-ceramic (HAC) has been proven extensively. For the reconstruction of juxta-articular cancellous bone defects with this synthetic material, the mechanical properties of the HAC-bone regeneration complex needed to be investigated. In order not to alter the specific ability of the articular structures to distribute and absorb loading stress, the physiological force-transmitting performance of the subchondral zone must be achieved by filling the defect within HAC. This study deals with the influence of a physiological load on the remodelling within HAC-filled subchondral bone defects. As orientation is the important factor affecting the physical properties of hard tissue, we show the morphological aspect of functional adaptation of the hydroxyapatite-bone compound determined by the orientation of the bone collagen fibres. By biomechanical methods, the elastic properties of the resulting ceramo-osseous regeneration complex were tested. Reproducible subchondral bone defects were prepared in medial femoral condyles of rabbits, leaving a 0.5 mm coplanar layer of bone and cartilage. The defects were filled with granules of HAC. Polarizing microscopy revealed the dynamical aspect of the bony integration of the material and the remodelling process under physiological locomotion. It showed a rapid ongrowth of collagen fibres on the ceramic surface. By its increasing orientation to domains from woven texture to economical trabecular architecture, the load-bearing facility is documented. Indenting the articular surface on an impressive force testing machine 18 months after HAC implantation proved the equal elastic response of the ceramo-osseous regeneration complex with the overlying structures in comparison with the integrity of not-operated femoral condyles. When integrated by bone, HAC fulfils in our dynamic animal model physiological demands even in large bone defects close to articular surfaces.On the occasion of his 60th birthday, we dedicate this study to Professor K. H. Jungbluth, Head of Trauma and Reconstructive Surgery Department, University Hospital of Hamburg.     

Characterization of neutrophil adhesion to different titanium surfaces

Although titanium (Ti) is known to elicit a foreign body response when implanted into humans, Ti implant healing resembles normal wound healing in terms of inflammatory cell recruitment and inflammation persistence. Rough implant surfaces may present better conditions for protein adsorption and for the adhesion of platelets and inflammatory cells such as neutrophils. Implanted biomedical devices initially interact with coagulating blood; however, direct contact between the oxide layer of the implant and neutrophils has not been completely described. The aim of the present study is to compare the behaviours of neutrophils in direct contact with different Ti surfaces. Isolated human neutrophils were placed into contact with Ti discs, which had been rendered as ‘smooth’ or ‘rough’, following different surface treatments. Scanning electron microscopy and flow cytometry were used to measure cell adhesion to the surfaces and exposure of membrane proteins such as CD62L and CD11b. Topographic roughness was demonstrated as higher for SLA treated surfaces, measured by atomic force microscopy and elemental analysis was performed by energy dispersive X-ray, showing a similar composition for both surfaces. The adhesion of neutrophils to the ‘rough’ Ti surface was initially stronger than adhesion to the ‘smooth’ surface. The cell morphology and adhesion marker results revealed clear signs of neutrophil activation by either surface, with different neutrophil morphological characteristics being observed between the two surface types. Understanding the cellular mechanisms regulating cell–implant interactions should help researchers to improve the surface topography of biomedical implant devices.