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.     

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.     

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.     

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.     

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.     

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.     

Matrix generation within a macroporous non-degradable implant for osteochondral defects is not enhanced with partial enzymatic digestion of the surrounding tissue: evaluation in an in vivo rabbit model

Articular cartilage defects are a significant source of pain, have limited ability to heal, and can lead to the development of osteoarthritis. However, a surgical solution is not available. To tackle this clinical problem, non-degradable implants capable of carrying mechanical load immediately after implantation and for the duration of implantation, while integrating with the host tissue, may be viable option. But integration between articular cartilage and non-degradable implants is not well studied. Our objective was to assess the in vivo performance of a novel macroporous, nondegradable, polyvinyl alcohol construct. We hypothesized that matrix generation within the implant would be enhanced with partial digestion of the edges of articular cartilage. Our hypothesis was tested by randomizing an osteochondral defect created in the trochlea of 14 New Zealand white rabbits to treatment with: (i) collagenase or (ii) saline, prior to insertion of the implant. At 1 and 3-month post-operatively, the gross morphology and histologic appearance of the implants and the surrounding tissue were assessed. At 3 months, the mechanical properties of the implant were also quantified. Overall, the hydrogel implants performed favorably; at all time-points and in all groups the implants remained well fixed, did not cause inflammation or synovitis, and did not cause extensive damage to the opposing articular cartilage. Regardless of treatment with saline or collagenase, at 1 month post-operatively implants from both groups had a contiguous interface with adjacent cartilage and were populated with chondrocyte-like cells. At 3 months fibrous encapsulation of all implants was evident, there was no difference between area of aggrecan staining in the collagenase versus saline groups, and implant modulus was similar in both groups; leading us to reject our hypothesis. In summary, a porous PVA osteochondral implant remained well fixed in a short term in vivo osteochondral defect model; however, matrix generation within the implant was not enhanced with partial digestion of adjacent articular cartilage.     

Recent Progress in Cartilage Tissue Engineering—Our Experience and Future Directions

Given the limited spontaneous repair that follows cartilage injury, demand is growing for tissue engineering approaches for cartilage regeneration. There are two major applications for tissue-engineered cartilage. One is in orthopedic surgery, in which the engineered cartilage is usually used to repair cartilage defects or loss in an articular joint or meniscus in order to restore the joint function. The other is for head and neck reconstruction, in which the engineered cartilage is usually applied to repair cartilage defects or loss in an auricle, trachea, nose, larynx, or eyelid. The challenges faced by the engineered cartilage for one application are quite different from those faced by the engineered cartilage for the other application. As a result, the emphases of the engineering strategies to generate cartilage are usually quite different for each application. The statuses of preclinical animal investigations and of the clinical translation of engineered cartilage are also at different levels for each application. The aim of this review is to provide an opinion piece on the challenges, current developments, and future directions for cartilage engineering for both applications.     

Engineering cartilage tissue interfaces using a natural glycosaminoglycan hydrogel matrix — An in vitro study

Hydrogels are suitable matrices for cartilage tissue engineering on account of their resemblance to native extracellular matrix of articular cartilage and also considering its ease of application, they can be delivered to the defect site in a minimally invasive manner. In this study, we evaluate the suitability of a fast gelling natural biopolymer hydrogel matrix for articular cartilage tissue engineering. A hydrogel based on two natural polymers, chitosan and hyaluronic acid derivative was prepared and physicochemically characterized. Chondrocytes were then encapsulated within the hydrogel and cultured over a period of one month. Cartilage regeneration was assessed by histological, biochemical and gene expression studies. Chondrocytes maintained typical round morphology throughout the course of this investigation, indicating preservation of their phenotype with sufficient production of extracellular matrix and expression of typical chondrogenic markers Collagen type 2 and aggrecan. The results suggest that the natural polymer hydrogel matrix can be used as an efficient matrix for articular cartilage tissue engineering.     

可降解水凝胶作为关节软骨修复材料的研究进展

可降解水凝胶因其良好的生物相容性和生物降解性被广泛用于关节软骨的修复和再生。本文以可降解水凝胶在软骨组织工程中的三类应用策略为主线,概述了用于原位成型可注射水凝胶的蛋白多糖类材料及纳米复合类材料;系统总结了传统工艺制造组织工程支架的优缺点及多种工艺结合的制备方法;重点归纳了近年来3D打印组织工程支架从纯软骨到骨/软骨一体化、从单层到多层的研究进展;最后分析了可降解水凝胶作为关节软骨支架材料在微观定向结构和生物活性功能化方面的局限性,并作出展望：未来开展多材料、多尺度、多诱导的高仿生梯度支架是关节软骨组织工程的一个重要研究方向。     

The growth of chondrocytes using Gelfoam® as a biodegradable scaffold

Successful articular cartilage resurfacing must overcome several problems: the implant must easily fit the defect, it must be stable within the defect before full incorporation of repair tissue has occurred, and the reparative tissue must closely approximate the structure of normal hyaline cartilage. To this end, several natural and synthetic components have been used, both in vivo and in vitro, to provide a scaffold. These include isolated chondrocyte allografts, intact cartilage allografts, periossteal grafts, reconstructed collagen sponges, hydrogels and carbon fibres. However, promising results have been reported using three dimensional scaffolds in culture with isolated chondrocytes with subsequent implantation. This preliminary in vitro study utilizes Gelfoam^® (a purified gelatin sponge) as such a scaffold. The biocompatibility of Gelfoam with both chondrocytes and osteoblast cells was first confirmed. The ability of chondrocytes to replicate and differentiate within Gelfoam scaffolds was assessed biochemically by measurement of the DNA content and glycosaminoglycans (GAG) production over 25 days in culture. The distribution of the cartilagenous matrix produced was observed by light microscopy, and the constituents of this matrix were assessed using specific antibodies and immunolocalization.     

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.     

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.     

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.     

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.     

Biofunctional polymer nanoparticles for intra-articular targeting and retention in cartilage

The extracellular matrix of dense, avascular tissues presents a barrier to entry for polymer-based therapeutics, such as drugs encapsulated within polymeric particles. Here, we present an approach by which polymer nanoparticles, sufficiently small to enter the matrix of the targeted tissue, here articular cartilage, are further modified with a biomolecular ligand for matrix binding. This combination of ultrasmall size and biomolecular binding converts the matrix from a barrier into a reservoir, resisting rapid release of the nanoparticles and clearance from the tissue site. Phage display of a peptide library was used to discover appropriate targeting ligands by biopanning on denuded cartilage. The ligand WYRGRL was selected in 94 of 96 clones sequenced after five rounds of biopanning and was demonstrated to bind to collagen II alpha1. Peptide-functionalized nanoparticles targeted articular cartilage up to 72-fold more than nanoparticles displaying a scrambled peptide sequence following intra-articular injection in the mouse.     

Formulation of PEG-based hydrogels affects tissue-engineered cartilage construct characteristics

The limited supply of cartilage tissue with appropriate sizes and shapes needed for reconstruction and repair has stimulated research in the area of hydrogels as scaffolds for cartilage tissue engineering. In this study we demonstrate that poly(ethylene glycol) (PEG)-based semi-interpenetrating (sIPN) network hydrogels, made with a crosslinkable poly(ethylene glycol)-dimethacrylate (PEGDM) component and a non-crosslinkable interpenetration poly(ethylene oxide) (PEO) component, and seeded with chondrocytes support cartilage construct growth having nominal thicknesses of 6 mm and relatively uniform safranin-O stained matrix when cultured statically, unlike constructs grown with prefabricated macroporous scaffolds. Even though changing the molecular weight of the PEO from 100 to 20 kDa reduces the viscosity of the precursor polymer solution, we have demonstrated that it does not appear to affect the histological or biochemical characteristics of cartilaginous constructs. Extracellular matrix (ECM) accumulation and the spatial uniformity of the ECM deposited by the embedded chondrocytes decreased, and hydrogel compressive properties increased, as the ratio of the PEGDM:PEO in the hydrogel formulation increased (from 30:70 to 100:0 PEGDM:PEO). Total collagen and glycosaminoglycan contents per dry weight were highest using the 30:70 PEGDM:PEO formulation (24.4+/-3.5% and 7.1+/-0.9%, respectively). The highest equilibrium compressive modulus was obtained using the 100:0 PEGDM:PEO formulation (0.32+/-0.07 MPa), which is similar to the compressive modulus of native articular cartilage. These results suggest that the versatility of PEG-based sIPN hydrogels makes them an attractive scaffold for tissue engineering of cartilage.     

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.     

A critical review on polymer-based bio-engineered materials for scaffold development

Since the last decade, tissue engineering has shown a sensational promise in providing more viable alternatives to surgical procedures for harvested tissues, implants and prostheses. Due to the fast development on biomaterial technologies, it is now possible for doctors to use patients’ cells to repair orthopedic defects such as focal articular cartilage lesions. In order to support the three-dimensional tissue formation, scaffolds made by biocompatible and bioresorbable polymers and composite materials, for providing temporary support of damaged body and cell structures have been developed recently. Although ceramic and metallic materials have been widely accepted for the development of implants, its non-resorbability and necessity of second surgical operation, which induces extra for the patients, limit their wide applications. This review article aims at introducing (i) concept of cartilage tissue engineering, (ii) common types of bio-engineered materials and (iii) future development of biomaterial scaffolds.     

Preparation and characterization of collagen/PLA,chitosan/PLA,and collagen/chitosan/PLA hybrid scaffolds for cartilage tissue engineering

In this study, three-dimensional (3D) porous scaffolds were developed for the repair of articular cartilage defects. Novel collagen/polylactide (PLA), chitosan/PLA, and collagen/chitosan/PLA hybrid scaffolds were fabricated by combining freeze-dried natural components and synthetic PLA mesh, where the 3D PLA mesh gives mechanical strength, and the natural polymers, collagen and/or chitosan, mimic the natural cartilage tissue environment of chondrocytes. In total, eight scaffold types were studied: four hybrid structures containing collagen and/or chitosan with PLA, and four parallel plain scaffolds with only collagen and/or chitosan. The potential of these types of scaffolds for cartilage tissue engineering applications were determined by the analysis of the microstructure, water uptake, mechanical strength, and the viability and attachment of adult bovine chondrocytes to the scaffolds. The manufacturing method used was found to be applicable for the manufacturing of hybrid scaffolds with highly porous 3D structures. All the hybrid scaffolds showed a highly porous structure with open pores throughout the scaffold. Collagen was found to bind water inside the structure in all collagen-containing scaffolds better than the chitosan-containing scaffolds, and the plain collagen scaffolds had the highest water absorption. The stiffness of the scaffold was improved by the hybrid structure compared to plain scaffolds. The cell viability and attachment was good in all scaffolds, however, the collagen hybrid scaffolds showed the best penetration of cells into the scaffold. Our results show that from the studied scaffolds the collagen/PLA hybrids are the most promising scaffolds from this group for cartilage tissue engineering.