Functionalized Hydrogels for Articular Cartilage Tissue Engineering

Articular cartilage(AC)is an avascular and flexible connective tissue located on the bone surface in the diarthrodial joints.AC defects are common in the knees of young and physically active individuals.Because of the lack of suitable tissue-engineered artificial matrices,current therapies for AC defects,espe-cially full-thickness AC defects and osteochondral interfaces,fail to replace or regenerate damaged carti-lage adequately.With rapid research and development advancements in AC tissue engineering(ACTE),functionalized hydrogels have emerged as promising cartilage matrix substitutes because of their favor-able biomechanical properties,water content,swelling ability,cytocompatibility,biodegradability,and lubricating behaviors.They can be rationally designed and conveniently tuned to simulate the extracel-lular matrix of cartilage.This article briefly introduces the composition,structure,and function of AC and its defects,followed by a comprehensive review of the exquisite(bio)design and(bio)fabrication of func-tionalized hydrogels for AC repair.Finally,we summarize the challenges encountered in functionalized hydrogel-based strategies for ACTE both in vivo and in vitro and the future directions for clinical translation.

Study on the Microstructure of Human Articular Cartilage/Bone Interface

For improving the theory of gradient microstructure of cartilage/bone interface, human distal femurs were studied. Scanning Electron Microscope （SEM）, histological sections and MicroCT were used to observe, measure and model the micro- structure of cartilage/bone interface. The results showed that the cartilage/bone interface is in a hierarchical structure which is composed of four different tissue layers. The interlocking of hyaline cartilage and calcified cartilage and that of calcified car- tilage and subchondral bone are in the manner of＂protrusion-pore＂ with average diameter of 17.0 gm and 34.1 lam respectively. In addition, the cancellous bone under the cartilage is also formed by four layer hierarchical structure, and the adjacent layers are connected by bone trabecula in the shape of H, I and Y, forming a complex interwoven network structure. Finally, the simplified structure model of the cartilage/bone interface was proposed according to the natural articular cartilage/bone interface. The simplified model is a 4-layer gradient biomimetic structure, which corresponds to four different tissues of natural cartilage/bone interface. The results of this work would be beneficial to the design of bionic scaffold for the tissue engineering of articular cartilage/bone.

Mesenchymal stem cells as a potent cell source for articular cartilage regeneration

Since articular cartilage possesses only a weak capac-ity for repair, its regeneration potential is considered one of the most important challenges for orthopedic surgeons. The treatment options, such as marrow stimulation techniques, fail to induce a repair tissue with the same functional and mechanical properties of native hyaline cartilage. Osteochondral transplantation is considered an effective treatment option but is as-sociated with some disadvantages, including donor-site morbidity, tissue supply limitation, unsuitable mechani-cal properties and thickness of the obtained tissue. Although autologous chondrocyte implantation results in reasonable repair, it requires a two-step surgical pro-cedure. Moreover, chondrocytes expanded in culture gradually undergo dedifferentiation, so lose morpho-logical features and specialized functions. In the search for alternative cells, scientists have found mesenchymal stem cells(MSCs) to be an appropriate cellular mate-rial for articular cartilage repair. These cells were origi-nally isolated from bone marrow samples and further investigations have revealed the presence of the cells in many other tissues. Furthermore, chondrogenic dif-ferentiation is an inherent property of MSCs noticedat the time of the cell discovery. MSCs are known to exhibit homing potential to the damaged site at which they differentiate into the tissue cells or secrete a wide spectrum of bioactive factors with regenerative proper-ties. Moreover, these cells possess a considerable im-munomodulatory potential that make them the general donor for therapeutic applications. All of these topics will be discussed in this review.

Benefits of Ilizarov automated bone distraction for nerves and articular cartilage in experimental leg lengthening

AIM To determine peculiarities of tissue responses to manual and automated Ilizarov bone distraction in nerves and articular cartilage.METHODS Twenty-nine dogs were divided in two experimental groups: Group M-leg lengthening with manual distraction(1 mm/d in 4 steps), Group A-automated distraction(1 mm/d in 60 steps) and intact group. Animals were euthanized at the end of distraction, at 30 th day of fixation in apparatus and 30 d after the fixator removal. M-responses in gastrocnemius and tibialis anterior muscles were recorded, numerical histology of peronealand tibialis nerves and knee cartilage semi-thin sections, scanning electron microscopy and X-ray electron probe microanalysis were performed.RESULTS Better restoration of M-response amplitudes in leg muscles was noted in A-group. Fibrosis of epineurium with adipocytes loss in peroneal nerve, subperineurial edema and fibrosis of endoneurium in some fascicles of both nerves were noted only in M-group, shares of nerve fibers with atrophic and degenerative changes were bigger in M-group than in A-group. At the end of experiment morphometric parameters of nerve fibers in peroneal nerve were comparable with intact nerve only in A-group. Quantitative parameters of articular cartilage(thickness, volumetric densities of chondrocytes, percentages of isogenic clusters and empty cellular lacunas, contents of sulfur and calcium) were badly changed in M-group and less changed in A-group.CONCLUSION Automated Ilizarov distraction is more safe method of orthopedic leg lengthening than manual distraction in points of nervous fibers survival and articular cartilage arthrotic changes.

A new method for computing the uniaxial modulus of articular cartilages using modified inhomogeneous triphasic model

It is well known that subtle changes in structure and tissue composition of articular cartilage can lead to its degeneration. The present paper puts forward a modified layered inhomogeneous triphasic model with four parameters based on the inhomogeneous triphasic model proposed by Narmoneva et al. Incorporating a piecewise fitting optimization criterion, the new model was used to obtain the uniaxial modulus Ha, and predict swelling pattern for the articular cartilage based on ultrasound-measured swelling strain data. The results show that the new method can be used to provide more accurate estimation on the uniaxial modulus than the inhomogeneous triphasic model with three parameters and the homogeneous mode, and predict effectively the swell- ing strains of highly nonuniform distribution of degenerated articular cartilages. This study can provide supplementary information for exploring mechanical and material properties of the cartilage, and thus be helpful for the diagnosis of osteoarthritis-related diseases.

Nanoparticle–Cartilage Interaction: Pathology-Based Intra-articular Drug Delivery for Osteoarthritis Therapy

Osteoarthritis is the most prevalent chronic and debilitating joint disease,resulting in huge medical and socioeconomic burdens.Intra-articular administration of agents is clinically used for pain management.However,the effectiveness is inapparent caused by the rapid clearance of agents.To overcome this issue,nanoparticles as delivery systems hold considerable promise for local control of the pharmacokinetics of therapeutic agents.Given the therapeutic programs are inseparable from pathological progress of osteoarthritis,an ideal delivery system should allow the release of therapeutic agents upon specific features of disorders.In this review,we firstly introduce the pathological features of osteoarthritis and the design concept for accurate localization within cartilage for sustained drug release.Then,we review the interactions of nanoparticles with cartilage microenvironment and the rational design.Furthermore,we highlight advances in the therapeutic schemes according to the pathology signals.Finally,armed with an updated understanding of the pathological mechanisms,we place an emphasis on the development of“smart”bioresponsive and multiple modality nanoparticles on the near horizon to interact with the pathological signals.We anticipate that the exploration of nanoparticles by balancing the efficacy,safety,and complexity will lay down a solid foundation tangible for clinical translation.

Effects of Structural Changes in Subchondral Bone on Articular Cartilage in a Beagle Dog Model

Objective Using MR T2-mapping and histopathologic score for articular cartilage to evaluate the effect of structural changes in subchondral bone on articular cartilage. Methods Twenty-four male Beagle dogs were randomly divided into a subchondral bone defect group （n = 12） and a bone cement group （n = 12）. Models of subchondral bone defectin the medial tibial plateau and subchondral bone filled with bone cement were constructed. In all dogs, the left knee joint was used as the experimental sideand the right knee as the sham side. The T2 value for articular cartilage at the medial tibial plateau was measured at postoperative weeks 4, 8, 16, and 24. The articular cartilage specimens were stained with hematoxylin and eosin, and evaluated using the Mankin score. Results There was a statistically significant difference （P 〈 0.05） in Mankin score between the bone defect group and the cement group at postoperative weeks 16 and 24. There was a statistically significant difference in the T2 values between the bone defect group and its sham group （P 〈 0.05） from week 8, and between the cement group and its sham group （P 〈 0.05） from week 16. There was significant difference in T2 values between the two experimental groups at postoperative week 24 （P 〈 0.01）. The T2 value for articular cartilage was positively correlated with the Mankin score （ρ = 0.758, P 〈 0.01）. Conclusion Structural changes in subchondral bone can lead to degeneration of the adjacent articular cartilage. Defects in subchondral bone cause more severe degeneration of cartilage than subchondral bone filled with cement. The T2 value for articular cartilage increases with the extent of degeneration. MR T2-mapping images and the T2 value for articular cartilage can indicate earlycartilage degeneration.

Elevated levels of interleukin-1β, interleukin-6, tumor necrosis factor-α and vascular endothelial growth factor in patients with knee articular cartilage injury

BACKGROUND Inflammatory cytokines play a vital role in the occurrence of osteoarticular injury and inflammation. Whether inflammation-associated factors interleukin-1β(IL- 1β), IL-6, tumor necrosis factor-α(TNF-α) and vascular endothelial growth factor (VEGF) are involved in the pathogenesis of keen articular cartilage injury remains poorly understood. AIM To measure the levels of inflammatory factors [IL-1β, IL-6, TNF-α and VEGF] in patients with knee articular cartilage injury. METHODS Fifty-five patients with knee articular cartilage injury were selected as patient groups, who were divided into three grades [mild (n = 20), moderate (n = 19) and severe (n = 16)] according to disease severity and X-ray examinations. Meanwhile, 30 healthy individuals who underwent physical examination were selected as the control group. The levels of IL-1β, IL-6, TNF-α and VEGF were measured by ELISA and immunohistochemical staining. RESULTS Compared with the control group, patient groups displayed significantly higher levels of IL-1β, IL-6, TNF-α and VEGF, and the extent of increase was directly proportional to the severity of injury (P < 0.05). In addition, the number of cells with positive staining of IL-1β, IL-6, TNF-α and VEGF in the synovial membrane were significantly increased, along with increased disease severity (P < 0.05). After treatment, the scores of visual analogue scale and the Western Ontario and McMaster University of Orthopaedic Index in patient groups were 2.26 ± 1.13 and 15.56 ± 7.12 points, respectively, which were significantly lower than those before treatment (6.98 ± 1.32 and 49.48 ± 8.96). Correlation analysis suggested that IL-1β and TNF-α were positively correlated with VEGF. CONCLUSION IL-1β, IL-6, TNF-α and VEGF levels are increased in patients with knee articular cartilage injury, and are associated with the disease severity, indicating they might play an important role in the occurrence and development of knee articular cartilage injury. Furthermore, therapeutically targeting them might be a novel approach for the treatment of keen articular cartilage injury.

The study on the mechanical characteristics of articular cartilage in simulated microgravity

The microgravity environment of a long-term space flight may induce acute changes in an astronaut＇s musculo-skeletal systems. This study explores the effects of simulated microgravity on the mechanical characteristics of articular cartilage. Six rats underwent tail suspension for 14 days and six additional rats were kept under normal earth gravity as controls. Swelling strains were measured using high-frequency ultrasound in all cartilage samples subject to osmotic loading. Site-specific swelling strain data were used in a triphasic theoretical model of cartilage swelling to determine the uniaxial modulus of the cartilage solid matrix. No severe surface irregularities were found in the cartilage samples obtained from the control or tail-suspended groups. For the tail-suspended group, the thickness of the cartilage at a specified site, as determined by ultrasound echo, showed a minor decrease. The uniaxial modulus of articular cartilage at the specified site decreased significantly, from （6.31 ± 3.37） MPa to （5.05 ± 2.98）MPa （p 〈 0.05）. The histology- stained image of a cartilage sample also showed a reduced number of chondrocytes and decreased degree of matrix staining. These results demonstrated that the 14 d simulated microgravity induced significant effects on the mechanical characteristics of articular cartilage. This study is the first attempt to explore the effects of simulated microgravity on the mechanical characteristics of articular cartilage using an osmotic loading method and a triphasic model. The conclusions may provide reference information for manned space flights and a better understanding of the effects of microgravity on the skeletal system.

The Frictional Coefficient of Bovine Knee Articular Cartilage

The normal displacement of articular cartilage was measured under load and in sliding, and the coefficient of friction during sliding was measured using a UMT-2 Multi-Specimen Test System. The maximum normal displacement under load and the start-up frictional coefficient have similar tendency of variation with loading time. The sliding speed does not significantly influence the frictional coefficient of articular cartilage.

Abnormal subchondral bone remodeling and its association with articular cartilage degradation in knees of type 2 diabetes patients

Type 2 diabetes （T2D） is associated with systemic abnormal bone remodeling and bone loss. Meanwhile, abnormal subchondral bone remodeling induces cartilage degradation, resulting in osteoarthritis （OA）. Accordingly, we investigated alterations in subchondral bone remodeling, microstructure and strength in knees from T2D patients and their association with cartilage degradation. Tibial plateaus were collected from knee OA patients undergoing total knee arthroplasty and divided into non-diabetic （n---70） and diabetes （n = 51） groups. Tibial plateaus were also collected from cadaver donors （n = 20） and used as controls. Subchondral bone microstructure was assessed using micro-computed tomography. Bone strength was evaluated by micro-finite-element analysis. Cartilage degradation was estimated using histology. The expression of tartrate-resistant acidic phosphatase （TRAP）, osterix, and osteocalcin were calculated using immunohistochemistry. Osteoarthritis Research Society International （OARSI） scores of lateral tibial plateau did not differ between non-diabetic and diabetes groups, while higher OARSI scores on medial side were detected in diabetes group. Lower bone volume fraction and trabecular number and higher structure model index were found on both sides in diabetes group. These microstructural alterations translated into lower elastic modulus in diabetes group. Moreover, diabetes group had a larger number of TRAP~ osteoclasts and lower number of Osterix~ osteoprogenitors and Osteocalcin~ osteoblasts. T2D knees are characterized by abnormal subchondral bone remodeling and microstructural and mechanical impairments, which were associated with exacerbated cartilage degradation. In regions with intact cartilage the underlying bone still had abnormal remodeling in diabetes group, suggesting that abnormal bone remodeling may contribute to the early pathogenesis of T2D-associated knee OA.

Osteoarthritis and Articular Cartilage: Biomechanics and Novel Treatment Paradigms

Background: Osteoarthritis is a widespread highly painful disabling age-related disease with no known cure. Although novel strategies for ameliorating osteoarthritic damage abound, it is likely that none will be successful over time if the entire spectrum of the disease and the effects of joint biomechanics on joint tissues are not carefully considered. Objectives: 1) To detail the structure of healthy articular cartilage, the key tissue affected by osteoarthritis. 2) To detail what aspects of cartilage damage best characterize osteoarthritis. 3) To consider the role of biomechanical factors in developing solutions to treat osteoarthritic joint damage. Methods: Literature sources from 1980 onwards that have contributed to our knowledge of the topics relevant to this paper were accessed and retrieved. The data were categorized into four predominant themes and conclusions about the state of our knowledge and future directives were formulated. Conclusions: Osteoarthritis prevalence remains high, and a cure appears elusive. A rich body of data has helped us to better understand the key tissue involved, and suggests a repair process might be feasible, if the basic collective information on the role of biomechanics in mediating or moderating articular cartilage integrity and function is forthcoming.

An analytical poroelastic model for laboratorial mechanical testing of the articular cartilage (AC)

The articular cartilage （AC） can be seen as a biphasic poroelastic material. The cartilage deformation under compression mainly leads to an interstitial fluid flow in the porous solid phase. In this paper, an analytical poroelastic model for the AC under laboratorial mechanical testing is developed. The solutions of interstitial fluid pressure and velocity are obtained. The results show the following facts. （i） Both the pressure and fluid velocity amplitudes are proportional to the strain loading amplitude. （ii） Both the amplitudes of pore fluid pressure and velocity in the AC depend more on the loading amplitude than on the frequency. Thus, in order to obtain the considerable fluid stimulus for the AC cell responses, the most effective way is to increase the loading amplitude rather than the frequency. （iii） Both the interstitiM fluid pressure and velocity are strongly affected by permeability variations. This model can be used in experimental tests of the parameters of AC or other poroelastic materials, and in research of mechanotransduction and injury mechanism involved interstitial fluid flow.

Serum Metabonomics of Articular Cartilage Destruction Induced by T-2 Toxin in Wistar Rats

The molecular pathogenesis of T-2 toxin-induced cartilage destruction has not been fully unraveled yet. The aim of this study was to detect changes in serum metabolites in a rat anomaly model with articular cartilage destruction. Thirty healthy male Wistar rats were fed a diet containing T-2 toxin （300 ng/kg chow） for 3 months. Histopathological changes in femorotibial cartilage were characterized in terms of chondrocyte degeneration/necrosis and superficial cartilage defect, and the endogenous metabolite profile of serum was determined by UPLC/Q-TOF MS. Treated rats showed extensive areas of chondrocyte necrosis and superficial cartilage defect in the articular cartilage. In addition, 8 metabolites were found to change significantly in these rats compared to the control group, including lyso PE （18：0/0：0）, lyso PC（14：0）, lyso PC[18：4 （6Z,9Z,12Z,15Z）], lyso PC[（16：1（9Z）], lyso PC（16：0）, L-valine, hippuric acid, and asparaginyl-glycine. These 8 metabolites associated with cartilage injury are mainly involved in phospholipid and amino acid metabolic pathways.

Direct Visualisation of the Depth-Dependent Mechanical Properties of Full-Thickness Articular Cartilage

Objective: The structural anisotropy of articular cartilage controls its deformation response. As proteoglycans and collagen vary with depth, simple uniaxial compression results in inhomogeneous deformation with distinct depth-dependent mechanical properties. Investigations into depth-dependent mechanical properties of articular cartilage have previously required tissue modification after specimen isolation. Such modifications include histological processes, freezing, subchondral bone removal, and fluorescent staining that may alter the tissue, limiting in vivo applicability. Design: Using a custom tissue-sectioning device, 0.1 mm thick unfixed, unstained, osetochondral samples were obtained. A customized apparatus loaded samples to 12.5%, 24%, and 29% compression in under a microscope with 10× magnification. Equilibrium load was measured after stress relaxation. Intra-tissue displacement was measured by tracing groups of cells between the different compression levels using a digital imaging program. Cell distance from the subchondral bone was measured to identify intratissue displacement and calculate strain. Results: The results reveal that stress levels and intratissue displacement increased with greater tissue compression (p p in vivo conditions and may provide an important method for analyzing the coordinated changes in cartilage composition and function due to ageing and disease.

Comparative study on identi¯cation of healthy and osteoarthritic articular cartilages by fourier transform infrared imaging and chemometrics methods

Two discriminant methods,partial least squares-discriminant analysis(PLS-DA)and Fisher's discriminant analysis(FDA),were combined with Fourier transform infrared imaging(FTIRI)to differentiate healthy and osteoarthritic articular cartilage in a canine model.Osteoarthritic cartilage had been developed for up to two years after the anterior cruciate ligament(ACL)transection in one knee.Cartilage specimens were sectioned into 10μm thickness for FTIRI.A PLS-DA model was developed after spectral pre-processing.All IR spectra extracted from FTIR images were calculated by PLS-DA with the discriminant accuracy of 90%.Prior to FDA,principal component analysis(PCA)was performed to decompose the IR spectral matrix into informative princi pal component matrices.Based on the different discriminant mechanism,the discriminant accuracy(96%)of PCA-FDA with high convenience was higher than that of PLS-DA.No healthy cartilage sample was mis assigned by these two methods.The above mentioned suggested that both integrated technologies of FTIRI-PLS-DA and,especially,FTIRI-PCA-FDA could become a promising tool for the discrimination of healthy and osteoarthritic cartilage specimen as well as the diagnosis of cartilage lesion at microscopic level.The results of the study would be helpful for better understanding the pathology of osteoarthritics.

Repair of articular cartilage defects in minipigs by microfracture surgery and BMSCs transplantation

Objective:To investigate the feasibility of minimal invasive repair of cartilage defect by arthroscope-aided microfracture surgery and autologous transplantation of mesenchymal stem cells. Methods: Bone marrow of minipigs was taken out and the bone marrow derived mesenchymal stem cells (BMSCs) were isolated and cultured to passage 3. Then 6 minipigs were randomly divided into 2 groups with 6 knees in each group. After the articular cartilage defect was induced in each knee, the left defect received microfracture surgery and was injected with 2.5 ml BMSCs cells at a concentration of 3×107 cells/ml into the articular cavity; while right knee got single microfracture or served as blank control group. The animals were killed at 8 or 16 weeks, and the repair tissue was histologically and immunohistochemically examined for the presence of type Ⅱ collagen and glycosaminoglycans (GAGs) at 8 and 16 weeks. Results: Eight weeks after the surgery, the overlying articular surface of the cartilage defect showed normal color and integrated to adjacent cartilage. And 16 weeks after surgery, hyaline cartilage was observed at the repairing tissues and immunostaining indicated the diffuse presence of this type Ⅱ collagen and GAGs throughout the repair cartilage in the treated defects. Single microfracture group had the repairing of fibrocartilage, while during the treatment, the defects of blank group were covered with fewer fiber tissues, and no blood capillary growth or any immunological rejection was observed. Conclusion: Microfracture technique and BMSCs transplantation to repair cartilage defect is characterized with minimal invasion and easy operation, and it will greatly promote the regeneration repair of articular cartilage defect.

A Novel Biomaterial for Cartilage Repair Generated by Self-Assembly: Creation of a Self-Organized Articular Cartilage-Like Tissue

Recently, attention has been drawn to tissue engineering and other novel techniques aimed at reconstruction of the joint. Regarding articular cartilage tissue engineering, three-dimensional materials created in vitro by cultivation of autologous chondrocytes or mesenchymal stem cells with a collagen gel have been implanted to replace defective parts of the articular cartilage in limited cases with the diseases such as trauma or arthritis. However, several passages of chondrocyte culture are required to obtain a sufficient number of cells for tissue engineering. Additionally, several other problems arise including dedifferentiation of chondrocytes during cell culture, which need to be solved from a viewpoint of cellular resources. The purpose of our study is to create a novel biomaterial possessing functions and structures comparable to native hyaline articular cartilage by utilizing the physicochemical properties of the cartilage matrix components themselves, in other words, employing a self-assembly technique instead of using chondrocytes to produce cartilage matrices eventually leading to articular cartilage tissue formation. We verified the conditions and accuracy of the self-organization process and analyzed the resulting micro structure using electron beam microscopy in order to study the technique involved in the self-organization which would be applicable to creation of cartilage-like tissue. We demonstrated that self-assembly of several cartilage components including type II collagen, proteoglycan and hyaluronic acid could construct self-assembled cartilage-like tissues characterized by nano composite structures comparable to human articular cartilage and by low friction coefficients as small as those of native cartilage.

Influence of bone morphogenetic protein on articular cartilage regeneration following periosteal grafting

Background: Autologous periosteal grafting is used as treatment for articular cartilage defect. Objective: To study the effect of bone morphogenetic protein (BMP) on articular cartilage regeneration following periosteal grafting. Methods: 16 healthy 15 week-old New Zealand white rabbits of both sexes (32 knees) were randomly divided into experimental group (group A) and control group (group B). A4.0 mmdiameter full-thickness articular cartilage defect was created in the femoral intercondylar fossa in all rabbits. Following this, a4.0 mmdiameter section of the periosteum was harvested from the anteromedial part of the upper tibial bone. In group A (eight rabbits, 16 knees), the cartilage defect was covered with periosteum, into which 20 μg BMP and 20% Pluronic were injected. In group B (eight rabbits, 16 knees), the cartilage defect was covered with periosteum, into which the same dosage of 0.9% NS (Normal saline) and 20% Pluronic were injected. All rabbits were sacrificed at 4, 8, and 12 weeks postoperatively, the cartilage defect areas were examined macroscopically and microscopically, and the morphology of the chondrocytes and collagen fibers were examined by scanning electron microscopy. Results: The filling of the defects with regenerated tissue was observed in both the group. The most notable improvement was that the cartilage regeneration in group A was obviously superior to that in group B, with the total histological score in group A significantly higher. Conclusion: BMP is an effective factor that could promote regeneration of articular cartilage and lead to successful cartilaginous resurfacing following periosteal