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.

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.

AAV-mediated expression of p65shRNA and bone morphogenetic protein 4 synergistically enhances chondrocyte regeneration

BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factorκB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen II synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.

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.

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.

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.

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.

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.

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.

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.

COMP ARISON OF MACROMOLECULAR COMPONENT DISTRIBUTIONS IN OSTEOARTHRITIC AND HEALTHY CARTILAGES BY FOURIER TRANSFORM INFRARED IMAGING

Fourier transform infrared imaging(FTIRI)was used to examine the depth-dependent content variations of macromolcular components,ollagen and protooglycan(PG),in osteoarthritic and healthy cartilages.Dried 6 pmm thick sections of canine knee cartilages were imaged at 6.25 pμrm pixel-size in FTIRI.By analyzing the infrared(IR)images and spectra,the depth dependence of characteristic band(sugar)intensity of PG show obvious difference bet ween the cartilage sections of(OA)and bealth.The result confimns that PG content decreases in the ostcoarthritic cartilage.However,no clear change occurs to collagen,suggesting that the OA influences little on the collagen content at early stage of OA.This observation will be helpful to further understand PG loss associated with pathological conditions in OA,and demonstrates that FIIRI has the po-tential to become an important analytical tool to identify early clinical signs of tissue degna-dation,such as PG loss even collagen disruption.

The effect of spinal cord injury on the expression of TGF-β and TNF-α in rat articular cartilage

Objective： To observe the expression of TGF-β and TNF-α in the spinal cord injured rat model and discuss the significance of the articular cartilage metabolism. Methods： 36 SD female rats were randomly divided into 2 groups： Rats models of spinal cord injury were implemented by Allen method. T10 laminectomy was performed in the control group. Both groups of rats were killed respectively in 1w, 3w and 6w. Hematoxylin-eosin stain was given to each slice in the model group and control group. Immunohistochemical stain was applied by using ABC method in the expression of TGF-β and TNF-α. Those expressed level were performed in image analysis and statistics process. Results： TGF-β and TNF-α were mainly distributed on the surface layer of the articular cartilage, with a weak expression in control group. The expression of TNF-α in the model group was more significant than that in the control group in the lw, and still remained an evident difference with that in control group until the 6w（P 〈 0.05）. TGF-β expression of the model group had no remarkable difference with the control group in the lw （P 〉 0.05） and prominently became stronger at 6w（P 〈 0.05）. Conclusion： The expression of TNF-α occurred early in the development of spinal cord injury, and the expression of TGF-β became stronger with the revival of spinal neural function. Both expressions were strengthened in articular cartilage in the 3rd week.

The finite element analysis of articular cartilage fiber-reinforced composite model under rolling load

Articular cartilage is a layer of low-friction,load-bearing soft hydrated tissue covering bone-ends in diarthrosis,which plays an important role in spreading the load,reducing the joint contact stress,joint friction and wear during exercise.The vital mechanical function

The Use of Bone Morphogenetic Protein-7 and Resveratrol in Collagen Type II of Articular Cartilage

This study aimed to investigate the effects of resveratrol and bone morphogenetic protein 7 on type II collagen from superficial and middle zone of porcine articular chondrocytes. Articular cartilage was isolated from dissected porcine knee joint n = 12. Isolated cells were plated as monolayers at a density of 1 × 105 cells/well in 12-well culture plates and incubated at 37℃ in a humid atmosphere of 5% carbon dioxide and 95% air. Cell cultures were treated for four days with various concentrations of bone morphogenetic protein-7 and resveratroL Cells were then collected and analysed for collagen type II expression by real time polymerase chain reaction and protein level quantification by enzyme-linked immunosorbent assay. Cartilage tissue sections were localised for collagen type II by immunohistochemistry. Moreover, resveratrol and bone morphogenetic protein-7 effects on cartilage matrix contents were analysed by histology. Resveratrol and bone morphogenetic protein-7 stimulates expression of collagen type II mRNA and protein level accumulation in the surface zone and middle zone at 50μM ＋ 300 ng/ml （RSV ＋ BMP-7）. Immunohistochemistry results confirmed the presence of collagen type II on articular cartilage. Histological tissue sections confirmed that chondrocytes were obtained from different zones of articular cartilage. The study suggests that a combination of bone morphogenetic protein-7 and resveratrol up-regulate the expression and synthesis of collagen type II.

Synthesis and Lubrication of Phosphorylcholine Derivative

The monomer of phosphorylcholine derivative, O-(5-（2-methacryloxy）-3, 3-dimethyl-3-azapentyl)-O’-（ω-hydroxy-octyl）-phosphatequaternary ammonium salt, was designed and synthesized successfully. It was characterized by the spectra ofHNMR and Mass spectra （ESI+）, and every signal was assigned. Then the lubricating characteristics of the phosphorylcholinederivative were investigated on the tribological setup of ball-oh-flat. The Ultra-High Molecular Weight Polyethylene（UHMWPE） flat was rotated against a stainless steel ball with 6 mm diameter. The load was 2.3 N, which corresponded to amaximal Hertz contact pressure of 29 MPa. Water, phosphorylcholine derivative, and Acrylic Acid （AA） solution were used aslubricants, respectively. Compared with AA, the phosphorylcholine derivative shows significant lubrication. It can be stronglyhydrated under water due to the charged segment in chemical structure. The thick water layers within the chains serves asboundary lubricants, and this is thought to be the molecular origins of lubricating behavior.

Expression of Transforming Growth Factor β_(1) in Mesenchymal Stem Cells: Potential Utility in Molecular Tissue Engineering for Osteochondral Repair

The feasibility of using gene therapy to treat full-thickness articular cartilage defects was investigated with respect to the transfection and expression of exogenous transforming growth factor(TGF)-β_(1)genes in bone marrow-derived mesenchymal stem cells(MSCs)in vitro.The full-length rat TGF-β_(1)cDNA was transfected to MSCs mediated by lipofectamine and then selected with G418,a synthetic neomycin analog.The transient and stable expression of TGF-β_(1)by MSCs was detected by using immunohistochemical staining.The lipofectamine-mediated gene therapy efficiently transfected MSCs in vitro with the TGF-β_(1)gene causing a marked up-regulation in TGF-β_(1)expression as compared with the vector-transfected control groups,and the increased expression persisted for at least 4 weeks after selected with G418.It was suggested that bone marrow-derived MSCs were susceptible to in vitro lipofectamine mediated TGF-β_(1)gene transfer and that transgene expression persisted for at least 4 weeks.Having successfully combined the existing techniques of tissue engineering with the novel possibilities offered by modern gene transfer technology,an innovative concept,i.e.molecular tissue engineering,are put forward for the first time.As a new branch of tissue engineering,it represents both a new area and an important trend in research.Using this technique,we have a new powerful tool with which:(1)to modify the functional biology of articular tissue repair along defined pathways of growth and differentiation and(2)to affect a better repair of full-thickness articular cartilage defects that occur as a result of injury and osteoarthritis.

Molecular Tissue Engineering: Applications for Modulation of Mesenchymal Stem Cells Proliferation by Transforming Growth Factor β_1 Gene Transfer

The effect of transforming growth factor β 1 (TGF β 1 ) gene transfection on the proliferation of bone marrow derived mesenchymal stem cells (MSC S ) and the mechanism was investigated to provide basis for accelerating articular cartilage repairing using molecular tissue engineering technology. TGF β 1 gene at different doses was transduced into the rat bone marrow derived MSCs to examine the effects of TGF β 1 gene transfection on MSCs DNA synthesis, cell cycle kinetics and the expression of proliferating cell nuclear antigen (PCNA). The results showed that 3 μl lipofectamine mediated 1 μg TGF β 1 gene transfection could effectively promote the proliferation of MSCs best; Under this condition (DNA/Lipofectamine=1μg/3μl), flow cytometry and immunohistochemical analyses revealed a significant increase in the 3 H incorporation, DNA content in S phase and the expression of PCNA. Transfection of gene encoding TGF β 1 could induce the cells at G0/G1 phase to S1 phase, modulate the replication of DNA through the enhancement of the PCNA expression, increase the content of DNA at S1 phase and promote the proliferation of MSCs. This new molecular tissue engineering approach could be of potential benefit to enhance the repair of damaged articular cartilage, especially those caused by degenerative joint diseases.