In vitro adhesion and biocompatability of osteoblast-like cells to poly(methylmethacrylate) and poly(ethylmethacrylate) bone cements

A bone cement, poly(ethylmethacrylate)/n-butylmethacrylate (PEMA/nBMA) has been developed with lower exotherm and monomer leaching compared to the traditional poly(methylmethacrylate)/methylmethacrylate (PMMA/MMA) cement. This study compares the in vitro biological response to the cements using primary human osteoblast-like cells (HOB). Cell attachment was qualified by immunolocalization of vinculin and actin cytoskeleton, showing more organization on PEMA/nBMA compared to PMMA/MMA. Proliferation was assessed using tritiated thymidine incorporation, and phenotype expression determined by measuring alkaline phosphatase (ALP) activity. An increase in proliferation and ALP activity was observed on PEMA/nBMA compared to PMMA/MMA. The results confirm the biocompatability of PEMA/nBMA, and an enhanced cell attachment and expression of differentiated cell phenotype.     

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 in vitro investigation of the PEMA/THFMA system using chondrocyte culture

Adult articular cartilage has a limited capacity for self-repair and a biomaterial to aid the process would be ideal. The polymer system, poly(ethyl methacrylate)/tetrahydrofurfuryl methacrylate (PEMA/THFMA) has shown potential in a rabbit model for such a role. The ability of the polymer system to support chondrocytes in vitro was investigated by light microscopy, transmission electron microscopy and biochemical assays. The PEMA/THFMA system maintained chondrocytes in agarose in a viable state with more glycosaminoglycan (GAG) produced per unit DNA after 14 days in culture compared to the tissue culture plastic control. Chondrocytes remained rounded on the polymer system surface as opposed to well spread on the Thermanox. The PEMA/THFMA system has been shown to be biocompatible for bovine chondrocytes maintaining them in a differentiated state with enhanced GAG production.     

Interactions of chondrocytes with methacrylate copolymers

Copolymers of poly(ethylmethacrylate) (PEMA) and tetrahydrofurfurylmethacrylate (THFMA) have been shown to exhibit potential as a biomaterial for use in cartilage repair. However, the interactions of chondrocytes with the polymer surface is not well understood. A series of novel methacrylate copolymers containing PEMA, THFMA and hydroxyethylmethacrylate (HEMA) were prepared and the ability of these various copolymers to support chondrocytes attachment in vitro has been assessed by the Alamar blue assay for cell number and environmental scanning electron microscopy (ESEM). As the mole fraction of HEMA in PEMA/THFMA/HEMA copolymers increased, chondrocyte attachment to the polymer surface in 24 h decreased. Chondrocytes maintained a rounded morphology and were strongly attached on the THFMA/PEMA polymer surface, but as the mole fraction of HEMA increased the cells present became much smaller with fewer cell to cell interactions. The effect of pre-adsorbing fibronectin on to the polymer surface on cell attachment was assessed both in the presence and absence of serum. Chondrocyte attachment was significantly reduced in serum-free medium. Pre-adsorption of fibronectin on to the copolymer surface substantially increased cell attachment in all cases. In conclusion, chondrocyte attachment and proliferation on these copolymers may be controlled by changes in the polymer surface chemistry and is highly sensitive to the presence of proteins either in the culture media or pre-adsorbed on to the copolymer surface. © 1998 Kluwer Academic Publishers     

A simple method of cartilage regeneration using a new polymerizing system: ultrastructural characteristics of the repair tissue

The ultrastructural characteristics of the repair tissue in large articular cartilage defects, filled with a heterocyclic polymerizing system were studied using transmission electron microscopy (TEM) and energy dispersive micro-analysis (EDMA). By six weeks post-implantation, the defects were resurfaced with predominantly hyaline-like articular cartilage. Chondrocytes in both the superficial and deep zones of the repair tissue were highly productive, secreting large amounts of proteoglycans, into a well-organized, rich in collagen fibrils, extracellular matrix. By contrast, in the repair tissue of the defects treated without the biomaterial, proteoglycan synthesis was less and the structure of the matrix was inferior. We conclude that the polymer enhances both chondrocyte metabolism and matrix organization, thus improving the quality of the repair tissue in articular cartilage defects.     

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.     

Porous methacrylate tissue engineering scaffolds: using carbon dioxide to control porosity and interconnectivity

Porous scaffold structures are used in tissue engineering to provide structural guidance for regenerating tissues. The use of carbon dioxide (CO₂) to create such scaffolds has received some attention in the past but many researchers believe that although CO₂ processing of polymers can lead to porous scaffolds there is limited interconnectivity between the pores. In this study, highly porous (greater than 85%) and well interconnected scaffolds were obtained in which the size, distribution and number of pores could be controlled. This control was achieved by altering the rate of venting from polymer discs saturated with CO₂ under modest temperature and pressure. The polymer used is a blend of poly (ethyl methacrylate) and tetrahydrofurfuryl methacrylate (PEMA/THFMA). This polymer system has shown promise for potential applications in cartilage repair.     

Mechanical properties of hydroxyapatite reinforced poly(ethylmethacrylate) bone cement after immersion in a physiological solution: influence of a silane coupling agent

PEMA–based bone cement has previously been shown to possess many advantages over traditional PMMA cements. One of these is the option of adding up to 40 wt % HA without a decrease in static mechanical strength, thus providing the potential for enhanced bioactivity. Bone cement, in vivo, is subjected to an aqueous environment and therefore, it is important to understand the influence of this upon the mechanical integrity of experimental cements. In this current investigation the static and dynamic properties of PEMA cement, with and without 30 wt % untreated and silanated HA, were examined after periods of immersion in Ringers solution. A commercial PMMA cement was also tested in a similar manner. Relatively small changes in static mechanical properties were observed after 12 weeks storage for the PEMA cements, the largest change being for the PEMA cement reinforced with silanated HA. The PMMA cement exhibited the largest change in static strength with a decrease of 16.6%. In contrast to these results, the fatigue properties of the PEMA cements were found to decrease significantly after storage in Ringers solution, again with the largest changes to the PEMA cement reinforced with silanated HA. This effect was attributed to the reduction in efficiency of the silane coupling agent in the presence of water. The fatigue resistance of the PMMA cement was not reduced after immersion in a saline environment.     

Bi-layered constructs based on poly(l-lactic acid) and starch for tissue engineering of osteochondral defects

Articular cartilage has a limited capacity to repair itself, and conventional therapeutic approaches have shown to have limited success as they are deficient and inconsistent in long-term repair. Tissue engineering has shown to be an alternative route to regenerate articular defects. In this work, new bi-layered scaffolds are developed in order to enhance the integration between the engineered cartilage tissue and the corresponding subchondral bone. The concept includes the use of a common polymer in both sides, poly(l-lactic acid), PLLA, to increase the bonding between them, and the use of compression moulding followed by particle leaching to process porous scaffolds with controllable porosities. A compact layer could be observed between the two layers that could be useful for independent cell culturing of the developed osteochondral constructs. A blend of starch and PLLA was used in the cartilage side, which was found to possess adequate hydration capability. For the bone region, where more stiffness and strength was required, PLLA reinforced with hydroxyapatite was used. Preliminary bioactivity tests demonstrated that the bone-layer could induce the formation of a calcium–phosphate layer in vitro, whereas the cartilage layer does not exhibit the ability for calcification.     

Combination of bone marrow concentrate and PGA scaffolds enhance bone marrow stimulation in rabbit articular cartilage repair

Bone marrow stimulation (BMS) has been regarded as a first-line procedure for the repair of articular cartilage. However, cartilage repair using BMS alone has so far not been ideal because cell homing to the required area has not been sufficient. The aim of this study was to investigate the feasibility of autologous bone marrow concentrate transplantation for the repair of large, full-thickness cartilage defects. Thirty rabbits were divided into five groups: untreated (control); BMS only (BMS); BMS followed by PGA implantation (PGA); BMS followed by a combination of PGA and autologous bone marrow concentrate (BMC); and BMS together with a composite of PGA and cultured bone marrow stem cells (BME). The animals were sacrificed at week 8 after operation, and HE staining, toluidine blue staining and immunohistochemistry were used to assess the repair of defects. The results showed that improved repair, including more newly formed cartilage tissue and hyaline cartilage-specific extracellular matrix, was observed in BMC group relative to the first three groups, in addition similar results were found between BMC and BME groups, however it took longer time for in vitro cell expansion in the BME group. This study demonstrates that the transplantation of autologous bone marrow concentrate is an easy, safe and potentially viable method to contribute to articular cartilage repair.     

Flexural and fatigue properties of a bone cement based upon polyethylmethacrylate and hydroxyapatite

Polymethylmethacrylate (PMMA) bone cement is commonly used in surgery to fix joint replacements into the bone. Although the operations are generally successful, loosening of the prosthesis does occur with fracture of the bone cement treated as the source of failure in some instances. Polyethylmethacrylate (PEMA) bone cement offers a promising alternative to PMMA due to its high ductility, low toxicity and low exotherm. In addition, hydroxyapatite (HA) particles can be added, while retaining the ductile properties of the material. In this study, the flexural and fatigue properties of this experimental cement, with and without HA reinforcement, have been examined. It was found that up to 40wt.% HA could be added with increases in both flexural strength and modulus. Specimens were subjected to tensiontension cyclic loading at a number of stress levels until catastrophic failure occurred. In comparison with a commercial PMMA cement, tested at relatively high stresses, the PEMA cement failed at lower cycles to failure. However, the data converged at the lower stresses employed which are closer to the physiological loading situation. With the addition of HA, although the cycles to failure were decreased, the deformation experienced by the PEMA-HA cement whilst being cycled was reduced.     

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.     

PMMA brush-containing two-solution bone cement: preparation, characterization, and influence of composition on cement properties

Two-solution bone cement consisting of poly (methyl methacrylate) (PMMA) brushes in methyl methacrylate has been developed as an alternative to the traditional two-solution (TSBC) and powder-liquid cements. It was hypothesized that the substitution of brushes, for the entire pre-polymer phase of the cement, would permit a decrease in solution viscosity at higher polymer fractions, and allow for physical entanglements with the cement matrix. Consequently, improved cement exothermal and mechanical properties could be expected with brush addition. PMMA brushes were grafted on the surface of cross-linked PMMA nanospheres following a multi-stage synthetic strategy. Brushes exhibiting optimal molecular weight for preparation of TSBC were used for characterization of cement viscosity, flexural and compressive mechanical properties, exothermal properties and residual monomer content. Interactions between grafts and free polymer formed during free radical polymerization of the cement were evaluated based on molecular weight measurements of the cement matrix and brushes. Brush-containing cements exhibited lower viscosity at significantly higher polymer fractions in comparison to TSBC. Cements with PMMA brushes had significantly lower polymerization temperatures and residual monomer content. Measurements of molecular weight revealed the existence of a dry brush regime when using the brush compositions selected in this study, which led to a reduction in the mechanical properties of some of the compositions tested. The optimal cement viscosity and maintenance of other important cement properties achieved with addition of PMMA brushes is expected to expand the use of the two-solution cements in a range of applications.     

Long term results after implantation of tissue engineered cartilage for the treatment of osteochondral lesions in a minipig model

In present study we determined the long term in vivo integration and histological modeling of an in vitro engineered cartilage construct. Tissue engineered autologous cartilagenous tissue was cultured on calcium phosphate cylinders and implanted into osteochondral defects into the femoral condyles in minipigs. Radiological follow-up was performed at 2, 8, 26 and 52 weeks, condyles were harvested 26 and 52 weeks post-implantation. Thickness of cultivated tissue (1.10 ± 0.55 mm) was comparable to in situ cartilage and cells produced in vitro cartilage specific proteins. In vivo, 26 and 52 weeks post-implantation defects were resurfaced with hyaline-like tissue, the implants were well integrated with no gap at the interface between the engineered neocartilage and the adjacent articular cartilage. Synthesis of type II collagen was detected 26 and 52 weeks after implantation. The modified ICRS score increased from 26 to 52 weeks. Histomorphometric evaluation revealed a decrease in cellularity in tissue engineered cartilage from 2.2-fold of native cartilage after 26 weeks to 1.5-fold after 52 weeks. In conclusion, these findings demonstrate the integration and maturation of tissue engineered cartilage pellets attached on a bone substitute carrier implanted in osteochondral defects over a long time. J. P. Petersen, P. Ueblacker, C. Goepfert have contributed equally to this study.     

Effects of glass ionomer cements on bone tissue

In vivo biocompatibility of glass ionomer cements (GICs) was evaluated for use in orthopaedic surgery using a rat model and compared with conventional bone cement, Polymethyl methacrylate, PMMA. The unset GICs and PMMA were inserted into the marrow cavities of rat femora and retained in situ for various periods of time. The PMMA bone cement showed complete biocompatibility with no interference with reparative bone. The conventional GIC with smaller glass particles and lower powder/liquid ratio showed an initial minor toxic effect on rat bone tissue with later disturbance of adjacent bone formation. The conventional GIC with larger-size glass particles and higher powder/liquid ratio and resin-modified GIC showed more severe toxic effect on rat tissue with the resin-modified GIC affecting the rat bone tissue later. The causes of toxicity associated with the conventional GIC with larger glass particles and higher powder/liquid ration and the resin-modified GIC are thought to be related with the unreacted acid component of both materials and longer ongoing metallic ion release.     

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.     

A silk fibroin/chitosan scaffold in combination with bone marrow-derived mesenchymal stem cells to repair cartilage defects in the rabbit knee

Bone marrow-derived mesenchymal stem cells (BMSCs) were seeded in a three-dimensional scaffold of silk fibroin (SF) and chitosan (CS) to repair cartilage defects in the rabbit knee. Totally 54 rabbits were randomly assigned to BMSCs + SF/CS scaffold, SF/CS scaffold and control groups. A cylindrical defect was created at the patellofemoral facet of the right knee of each rabbit and repaired by scaffold respectively. Samples were prepared at 4, 8 and 12 weeks post-surgery for gross observation, hematoxylin–eosin and toluidine blue staining, type II collagen immunohistochemistry, Wakitani histology. The results showed that differentiated BMSCs proliferated well in the scaffold. In the BMSCs + SF/CS scaffold group, the bone defect was nearly repaired, the scaffold was absorbed and immunohistochemistry was positive. In the SF/CS scaffold alone group, fiber-like tissues were observed, the scaffold was nearly degraded and immunohistochemistry was weakly positive. In the control group, the defect was not well repaired and positive immunoreactions were not detected. Modified Wakitani scores were superior in the BMSCs + SF/CS scaffold group compared with those in other groups at 4, 8 and 12 weeks (P < 0.05). A SF/CS scaffold can serve as carrier for stem cells to repair cartilage defects and may be used for cartilage tissue engineering.     

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.     

Bone engineering on the basis of periosteal cells cultured in polymer fleeces

Large bone defects caused by severe trauma, infection or tumor resection are still a major challenge for orthopaedic surgery. The key concept for successful bone regeneration consists of combining the osteoinductive effect of osteogenic cells with a suitable carrier structure to promote osteoblastic differentiation and optimal matrix production. Therefore, periosteal cells cultured in polyglycolic-polylactid acid (PGLA) fleeces were investigated for their osteogenic differentiation and used to repair critical size bone defects in a rabbit model. Periosteal cells were isolated from New Zealand White rabbits and expanded in vitro. Osteogenic differentiation was investigated by analysis of alkaline phosphatase and osteocalcin production in vitro depending on culture conditions and passage number. Cells were seeded into PGLA fleeces. After further cultivation, tissue constructs were examined histologically and by immunohistochemistry for cell distribution and osteogenic differentiation. These constructs of defined size were used to repair critical size calvarial defects (group I) in rabbits compared to a defect repair with polymers only (group II) or to untreated defects (group III). Bone healing was evaluated after 4 weeks by radiodensitometry and a special histological scoring system. For early evaluation, radiodensitometry was not sensitive enough to detect differences in calcification. However, on histologic examination the group with cell/fleece constructs revealed intense formation of uncalcified bone. The mean defect closure of the experimental group I was 65%, compared to control groups II and III with 31% and 22%, respectively. The established methods of 3-D-cell culture and ex-vivo transplant assessment proved to be a valuable tool for quality assurance. The results demonstrate that the combination of periosteal cells and polymer fleeces is a tissue engineering approach, which may have clinical applications in various fields of reconstructive surgery.     

Multifunctional chondroitin sulphate for cartilage tissue-biomaterial integration

A biologically active, high-strength tissue adhesive is needed for numerous medical applications in tissue engineering and regenerative medicine. Integration of biomaterials or implants with surrounding native tissue is crucial for both immediate functionality and long-term performance of the tissue. Here, we use the biopolymer chondroitin sulphate (CS), one of the major components of cartilage extracellular matrix, to develop a novel bioadhesive that is readily applied and acts quickly. CS was chemically functionalized with methacrylate and aldehyde groups on the polysaccharide backbone to chemically bridge biomaterials and tissue proteins via a twofold covalent link. Three-dimensional hydrogels (with and without cells) bonded to articular cartilage defects. In in vitro and in vivo functional studies this approach led to mechanical stability of the hydrogel and tissue repair in cartilage defects.