Objectives. The aim of this study was to investigate the effect of granulocyte-colony stimulating factor (G-CSF) on mesenchymal stem cell (MSC) proliferation in vitro and to determine whether pre-microfracture systemic administration of G-CSF (a bone marrow stimulant) could improve the quality of repaired tissue of a full-thickness cartilage defect in a rabbit model. Methods. MSCs from rabbits were cultured in a control medium and medium with G-CSF (low-dose: 4 μg, high-dose: 40 μg). At one, three, and five days after culturing, cells were counted. Differential potential of cultured cells were examined by stimulating them with a osteogenic, adipogenic and chondrogenic medium. A total of 30 rabbits were divided into three groups. The low-dose group (n = 10) received 10 μg/kg of G-CSF daily, the high-dose group (n = 10) received 50 μg/kg daily by subcutaneous injection for three days prior to creating cartilage defects. The control group (n = 10) was administered saline for three days. At 48 hours after the first injection, a 5.2 mm diameter cylindrical osteochondral defect was created in the femoral trochlea. At four and 12 weeks post-operatively, repaired tissue was evaluated macroscopically and microscopically. Results. The cell count in the low-dose G-CSF medium was significantly higher than that in the control medium. The differentiation potential of MSCs was preserved after culturing them with G-CSF. Macroscopically, defects were filled and surfaces were smoother in the G-CSF groups than in the control group at four weeks. At 12 weeks, the quality of repaired cartilage improved further, and defects were almost completely filled in all groups. Microscopically, at four weeks, defects were partially filled with hyaline-like cartilage in the G-CSF groups. At 12 weeks, defects were repaired with hyaline-like cartilage in all groups. Conclusions. G-CSF promoted proliferation of MSCs in vitro. The systemic administration of G-CSF promoted the repair of damaged cartilage possibly through increasing the number of MSCs in a rabbit model. Cite this article: T. Sasaki, R. Akagi, Y. Akatsu, T. Fukawa, H. Hoshi, Y. Yamamoto, T. Enomoto, Y. Sato, R. Nakagawa, K. Takahashi, S. Yamaguchi, T. Sasho. The effect of systemic administration of G-CSF on a full-thickness cartilage defect in a rabbit model MSC proliferation as presumed mechanism: G-CSF for cartilage repair. Bone Joint Res 2017;6:123–131. DOI: 10.1302/2046-3758.63.BJR-2016-0083

Objectives. Previously, we reported the improved transfection efficiency of a plasmid DNA-chitosan (pDNA-CS) complex using a phosphorylatable nuclear localization signal-linked nucleic kinase substrate short peptide (pNNS) conjugated to chitosan (pNNS-CS). This study investigated the effects of pNNS-CS-mediated miR-140 and interleukin-1 receptor antagonist protein (IL-1Ra) gene transfection both in rabbit chondrocytes and a cartilage defect model. Methods. The pBudCE4.1-miR-140, pBudCE4.1-IL-1Ra, and negative control pBudCE4.1 plasmids were constructed and combined with pNNS-CS to form pDNA/pNNS-CS complexes. These complexes were transfected into chondrocytes or injected into the knee joint cavity. Results. High IL-1Ra and miR-140 expression levels were detected both in vitro and in vivo. In vitro, compared with the pBudCE4.1 group, the transgenic group presented with significantly increased chondrocyte proliferation and glycosaminoglycan (GAG) synthesis, as well as increased collagen type II alpha 1 chain (COL2A1), aggrecan (ACAN), and TIMP metallopeptidase inhibitor 1 (TIMP-1) levels. Nitric oxide (NO) synthesis was reduced, as were a disintegrin and metalloproteinase with thrombospondin type 1 motif 5 (ADAMTS-5) and matrix metalloproteinase (MMP)-13 levels. In vivo, the exogenous genes reduced the synovial fluid GAG and NO concentrations and the ADAMTS-5 and MMP-13 levels in cartilage. In contrast, COL2A1, ACAN, and TIMP-1 levels were increased, and the cartilage Mankin score was decreased in the transgenic group compared with the pBudCE4.1 group. Double gene combination produced greater efficacies than each single gene, both in vitro and in vivo. Conclusion. This study suggests that pNNS-CS is a good candidate for treating cartilage defects via gene therapy, and that IL-1Ra in combination with miR-140 produces promising biological effects on cartilage defects. Cite this article: R. Zhao, S. Wang, L. Jia, Q. Li, J. Qiao, X. Peng. Interleukin-1 receptor antagonist protein (IL-1Ra) and miR-140 overexpression via pNNS-conjugated chitosan-mediated gene transfer enhances the repair of full-thickness cartilage defects in a rabbit model. Bone Joint Res 2019;8:165–178. DOI: 10.1302/2046-3758.83.BJR-2018-0222.R1

Objectives. The lack of effective treatment for cartilage defects has prompted investigations using tissue engineering techniques for their regeneration and repair. The success of tissue-engineered repair of cartilage may depend on the rapid and efficient adhesion of transplanted cells to a scaffold. Our aim in this study was to repair full-thickness defects in articular cartilage in the weight-bearing area of a porcine model, and to investigate whether the CD44 monoclonal antibody biotin-avidin (CBA) binding technique could provide satisfactory tissue-engineered cartilage. Methods. Cartilage defects were created in the load-bearing region of the lateral femoral condyle of mini-type pigs. The defects were repaired with traditional tissue-engineered cartilage, tissue-engineered cartilage constructed with the biotin-avidin (BA) technique, tissue-engineered cartilage constructed with the CBA technique and with autologous cartilage. The biomechanical properties, Western blot assay, histological findings and immunohistochemical staining were explored. Results. The CBA group showed similar results to the autologous group in biomechanical properties, Moran’s criteria, histological tests and Wakitani histological scoring. Conclusions. These results suggest that tissue-engineered cartilage constructed using the CBA technique could be used effectively to repair cartilage defects in the weight-bearing area of joints. Cite this article: H. Lin, J. Zhou, L. Cao, H. R. Wang, J. Dong, Z. R. Chen. Tissue-engineered cartilage constructed by a biotin-conjugated anti-CD44 avidin binding technique for the repairing of cartilage defects in the weight-bearing area of knee joints in pigs. Bone Joint Res 2017;6:–295. DOI: 10.1302/2046-3758.65.BJR-2016-0277

Introduction: Within the last few years numerous operative procedures have been described aiming a biological repair of damaged articular cartilage. Current techniques are: Microfracture, Osteochondral Autografting (Mosaicplasty) and Autologous Chondrocyte Transplantation (ACT). Several new studies have shown, that the defect size plays a major role in the clinical outcome of the different procedures. Thus, it makes sense to measure the size of a cartilage defect before indicating a certain method for biological repair. Material and Methods: We have developed a software (beta-version) for measuring the size of a cartilage defect during a routine arthroscopy in a real-time mode. The programme is based on an Infrared-Navigation tool (Orthopilot, B.Braun-Aesculap, Germany). In order to proof the reliability and the usefulness of this device, we carried out following study: in each of 6 cadaver-knees we performed 2 full-thickness cartilage defects (MFC and LFC) of different size. In a first run 3 surgeons had to scope the joint and estimate the defect size with means of a scaled probe-hook. In a second run we performed a measurement of the defect with the Orthopilot™; finally an open measurement after arthrotomy was done to act as reference. Results: Measurement of the cartilage defect size was clearly superior to an estimation by probehook. Especially the inter-observer difference between the surgeons was widely spread, whereas the max. mismeasurement with the Orthopilot was 2mm. Discussion: Our study has shown, that navigational-assisted determination of chondral defects is superior to a simple estimation of a defect size by a probehook. Considering that the defect size is a crucial point in choosing the appropriate procedure for the treatment of cartilage defects, navigation devices like the CDM-software is maybe a helpful tool in making the right decision for a suitable method of biological cartilage repair

Aims

The present study investigates the effectiveness of platelet-rich plasma (PRP) gel without adjunct to induce cartilage regeneration in large osteochondral defects in a rabbit model.

In an experimental study in rabbits, bone and cartilage regeneration could be achieved with a new class of resorbable bio-implants. These implants consist of an open porous structure made from polylacitdes and an open porous fleece made from polyglactin/polydioxanon. Both layers were not separated from each other, thus allowing mesenchymal cells to penetrate freely from bone into both the bone substitute and the cartilage substitute layer. It could be shown that ostochondral defects of 4mm diameter and 6mm depth in the condyle of the knee of rabbits healed by the process of mesenchymal cell differentiation into osteocytes and chondrocytes triggered by mechanical load induction only. Evaluation of the newly formed cartilage by light microscopy and immunohistology showed hyaline like features. However, in many clinical cases chondral defects occur without substantial accompanying bone loss. In these situations, reconstruction of the cartilage defects only seems to be sufficient. However, fixation of such fleeces onto the bone is difficult. On one hand, adherence of the fleece to the underlying bone is crucial, on the other hand an open connection from the bone to the fleece must be accomplished in order to allow mesenchymal cells to penetrate the fleece. Therefor, any kind of glue fixation is not appropriate. To overcome this problem, a new fixation method was developed which allows a safe connection of the fleece onto the bone while providing an open contact of the fleece to the bone marrow for unhampered migration of mesenchymal cells. The new “Cartilage patches” consist of a fleece (serving as the cartilage substitute layer) made from polyglactin/polydioxanon which had proven its applicability in the above mentioned experiments. Fixation of fleece was achieved by “darts” which were glued onto the fleece. The darts were made from polylacitdes, thus providing sufficient mechanical stability in the bone. During operation, small holes are cut into the bone by a special instrument. The holes are located in such a way that the darts of the cartilage patch fit into them, such resulting in a stable fixation of the fleece onto the underlying bone. Blood containing mesenchymal cells from the bone marrow is able to flow from the holes into the fleece. In a biomechanical analysis the adherence of the cartilage patches were tested with respect to shear resistance and pull-out stabillity. The results of the tests show that the new cartilage patches withstand the mechanical stress exerted onto articular surfaces and can serve as a new class of cartilage substitute layers. In an animal experiment the applicability of the cartilage patches in reconstruction of cartilage defects in the knee joint of sheep will be proven

Transforming growth factor-beta2 (TGF-β2) is recognized as a versatile cytokine that plays a vital role in regulation of joint development, homeostasis, and diseases, but its role as a biological mechanism is understood far less than that of its counterpart, TGF-β1. Cartilage as a load-resisting structure in vertebrates however displays a fragile performance when any tissue disturbance occurs, due to its lack of blood vessels, nerves, and lymphatics. Recent reports have indicated that TGF-β2 is involved in the physiological processes of chondrocytes such as proliferation, differentiation, migration, and apoptosis, and the pathological progress of cartilage such as osteoarthritis (OA) and rheumatoid arthritis (RA). TGF-β2 also shows its potent capacity in the repair of cartilage defects by recruiting autologous mesenchymal stem cells and promoting secretion of other growth factor clusters. In addition, some pioneering studies have already considered it as a potential target in the treatment of OA and RA. This article aims to summarize the current progress of TGF-β2 in cartilage development and diseases, which might provide new cues for remodelling of cartilage defect and intervention of cartilage diseases

Abstract. Background. Recurrent patellar dislocation in combination with cartilage injures are difficult injuries to treat with confounding pathways of treatment. The aim of this study is to compare the clinical and functional outcomes of patients operated for patellofemoral instability with and without cartilage defects. Methods. 82 patients (mean age-28.8 years) with recurrent patellar dislocations, who underwent soft-tissue or bony procedures, were divided into 2 matched groups (age, sex, follow-up and type of procedure) of 41 each based on the presence or absence of cartilage defects in patella. Chondroplasty, microfracture, osteochondral fixation or AMIC-type procedures were done depending on the nature of cartilage injury. Lysholm, Kujala, Tegner and Subjective Knee scores of both groups were compared and analysed. Complications and return to theatre were noted. Results. With a mean follow-up of 8 years (2 years-12.3 years), there was a significant improvement observed in all the mean post-operative Patient Reported Outcome Measures (p<0.05) of both the groups, as compared to the pre-operative scores. Comparing the 2 groups, post-operative Lysholm, Kujala and Subjective knee scores were significantly higher in patients operated without cartilage defects (p<0.05). 3 patients operated for PFJ instability with cartilage defects had to undergo patellofemoral replacement in the long term. Odds ratio for developing complications is 2.6 for patients operated with cartilage defects. Conclusion. Although there is a significant improvement in the long term outcome scores of patients operated for recurrent patellar dislocation with cartilage defects, the results are significantly inferior as compared to those without cartilage defects, along with a higher risk of developing complications and returning to theatre. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Background. Recurrent patellar dislocation in combination with cartilage injures are difficult injuries to treat with confounding pathways of treatment. The aim of this study is to compare the clinical and functional outcomes of patients operated for patellofemoral instability with and without cartilage defects. Methods. 82 patients (mean age-28.8 years) with recurrent patellar dislocations, who underwent soft-tissue or bony procedures, were divided into 2 matched groups (age, sex, follow-up and type of procedure) of 41 each based on the presence or absence of cartilage defects in patella. Chondroplasty, microfracture, osteochondral fixation or Autologous Matrix-Induced Chondrogenesis(AMIC)-type procedures were done depending on the nature of cartilage injury. Lysholm, Kujala, Tegner and Subjective Knee scores of both groups were compared and analysed. Complications and return to theatre were noted. Results. With a mean follow-up of 8 years (2 years-12.3 years), there was a significant improvement observed in all the mean post-operative Patient Reported Outcome Measures (p<0.05) of both the groups, as compared to the pre-operative scores. Comparing the 2 groups, post-operative Lysholm, Kujala and Subjective knee scores were significantly higher in patients operated without cartilage defects (p<0.05). 3 patients operated for patellofemoral instability with cartilage defects had to undergo patellofemoral replacement in the long term. Odds ratio for developing complications is 2.6 for patients operated with cartilage defects. Conclusion. Although there is a significant improvement in the long term outcome scores of patients operated for recurrent patellar dislocation with cartilage defects, the results are significantly inferior as compared to those without cartilage defects, along with a higher risk of developing complications and returning to theatre

Abstract. Objectives. Little is known about the impact of cartilage defects on knee joint biomechanics. This investigation aimed to determine the gait characteristics of patients with symptomatic articular cartilage lesions of the knee. Methods. Gait analyses were performed at the Regional North-West Joint Preservation Centre. Anthropometric measurements were obtained, then 16 retroreflective markers representing the Plug-in-Gait biomechanical model were placed on pre-defined anatomical landmarks. Participants walked for two minutes at a self-selected speed on a treadmill on a level surface, then for 2 minutes downhill. A 15-camera motion-capture system recorded the data. Knee kinematics were exported into Matlab to calculate the average kinematics and spatiotemporal parameters per patient across 20 gait cycles. Depending on the normality of the data, paired t-tests or Wilcoxon ranked tests were performed to compare both knees (α = 0.05). Results. 20 patients participated; one of whom has bilateral cartilage defects. All 20 data sets were analysed for level walking; 18 were analysed for downhill walking. On a level surface, patients walked at an average speed of 3.1±0.8km/h with a cadence of 65.5±15.3 steps/minute. Patients also exhibited equal step lengths (0.470±0.072m vs 0.471±0.070m: p=0.806). Downhill, the average walking speed was 2.85±0.5km/h with a cadence of 78.8±23.1 steps/minute and step lengths were comparable (0.416±0.09m vs 0.420±0.079m: p=0.498). During level walking, maximum flexion achieved during swing did not differ between knees (54.3±8.6° vs 55.5±11.0°:p=0.549). Neither did maximal extension achieved at heel strike (3.1±5.7° vs 5.4±4.7°:p=0.135). On average, both knees remained in adduction throughout the gait cycle, with the degree of adduction greater in flexion in the operative knee. However, differences in maximal adduction were not significant (22.4±12.4° vs 18.7±11.0°:p=0.307). Maximal internal-external rotation patterns were comparable in stance (0.9±7.7° vs 3.5±9.8°: p=0.322) and swing (7.7±10.9° vs 9.8±8.3°:p=0.384). During downhill walking, maximum flexion also did not differ between operative and contralateral knees (55.38±10.6° vs 55.12±11.5°:p=0.862), nor did maximum extension at heel strike (1.32±6.5° vs 2.73±4.5°:p=0.292). No significant difference was found between maximum adduction of both knees (15.87±11.0° vs 16.78±12.0°:p=0.767). In stance, differences in maximum internal-external rotation between knees were not significant (5.39±10.7° vs 6.10±11.8°:p=0.836), nor were they significant in swing (7.69±13.3° vs 7.54±8.81°:p=0.963). Conclusions. Knee kinematics during level and downhill walking were symmetrical in patients with a cartilage defect of the knee, but an increased adduction during flexion in the operative knee may lead to pathological loading across the medial compartment of the knee during high flexion activities. Future work will investigate this further and compare the data to a healthy young population. We will also objectively assess the functional outcome of this joint preservation surgery to monitor its success. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Osteoarthritis (OA) affects bone cartilage and underlying bone. Mechanically, the underlying bone provides support to the healthy growth of the overlying cartilage. However, with the progress of OA, bone losses and cysts occur in the bone and these would alter the biomechanical behaviour of the joint, and further leading to bone remodelling adversely affect the overlying cartilage. Human femoral head and femoral condyle were collected during hip or knee replacement operation due to the end stage of osteoarthritis (age 50–70), and the cartilage patches were graded and marked. A volunteer patient, with minor cartilage injury in his left knee while the right knee is intact, was used as control. Peripheral quantitative computed tomography (pQCT) was used to scan the bone and to determine the volumetric bone mineral density (vBMD) distribution. The examination of retrieved tissue explants from osteoarthritic patients revealed that patches of cartilage were worn away from the articular surface, and patches of intact cartilage were left. The cysts, ranging from 1 to 10mm were existed in all osteoarthritic bones, and were located close to cartilage defects in the weight-bearing regions, and closely associated with the grade of cartilage defect as measured by pQCT. The bone mineral density (vBMD) distribution demonstrated that the bones around cysts had much higher vBMD than the trabecular bone away from the cysts. Compared to the subchondral bone under thicker cartilage, subchondral bone within cartilage defect has higher vBMD. This may result from the mechanical stimulation as a result of bone-bone direct contact with less protection of cartilage in cartilage defect regions. This study showed an association between cartilage defect and subchondral bone mineral density distribution. Cysts were observed in all osteoarthritic samples and they are located close to cartilage defects in the weight-bearing regions. Cartilage defect altered the loading pattern of the joints, this leading to the bone remodelling and resultant bone structural changes as compared to the normal bone tissues. This work was financially supported by The ARUK Proof of Concept Award (grant no: 21160)

Osteoarthritis (OA), the most prevalent chronic joint disease, represents a relevant social and economic burden worldwide. Human umbilical cord mesenchymal stem cells (HUCMSCs) have been used for injection into the joint cavity to treat OA. The aim of this article is to clarify whether Huc-MSCs derived exosomes could inhibit the progression of OA and the mechanism in this process. A rabbit OA model was established by the transection of the anterior cruciate ligament. The effects of HUCMSCs or exosomes derived from HUCMSCs on repairing articular cartilage of knee osteoarthritis was examined by micro-CT. Immunohistochemical experiments were used to confirm the expression of relevant inflammatory molecules in OA. In vitro experiments, Transwell assay was used to assess the migration of macrophages induced by TNF-a. Results showed that a large number of macrophages migrated in arthcular cavity in OA model in vivo, while local injection of HUCMSCs and exosomes did repair the articular cartilage. Immunohistochemical results suggested that the expression of CCL2 and CD68 in the OA rabbit model increased significantly, but was significantly reduced by HUCMSCs or exosomes. Transwell assay showed that both HUCMSCs and exosomes can effectively inhibit the migration of macrophage. In conclusion, the exosomes derived by HUCMSCs might might rescue cartilage defects in rabbit through its anti-inflammatory effects through inhibiting CCL2

Femoroacetabular impingement is a prearthritic deformity frequently associated with early chondral damage. Several techniques exist for restoring larger cartilage defects. While AMIC proved to be an effective treatment in knee and ankle, there are only short-term data available in hip. This study aimed to investigate the mid-term clinical outcome of patients with chondral lesions treated by AMIC and evaluate the quality of repair tissue via MRI. This retrospective, single center study includes 18 patients undergoing surgical hip dislocation for FAI between 2013 and 2016. Inclusion criteria were: cam or pincer-type FAI, femoral or acetabular chondral lesions > 1 cm. 2. , (IRCS III-IV). Due to exclusion criteria and loss-to-follow-up 9 patients (10 hips) could be included. Patient reported outcome measures included Oxford Hip Score (OHS) & Core Outcome Measure Index (COMI)). MRIs were evaluated using the Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) score. None of the patients underwent revision surgery except screw removals from the greater trochanter. Followup data indicate a satisfactory to good hip function at 5 years: PROMS improved from pre- to postop at 5 years: OHS from 38.1 to 43.4, COMI from to 1.8 and UCLA from 4 to 8.1 respectively. MOCART score was 67.5 postoperatively. Subgrouping showed slightly better results for acetabular defects (Ø 69.4) compared femoral defects (Ø 60). Based on the reported mid-term results, we consider AMIC as a valuable treatment option for larger chondral defects of the hip

Abstract. Background. Osteochondral allograft (OCA) transplantation is a clinically and cost-effective option for symptomatic cartilage defects. In 2017 we initiated a program for OCA transplantation for complex chondral and osteochondral defects as a UK tertiary referral centre. Aim. To characterise the complications, re-operation rate, graft survivorship and clinical outcomes of knee OCA transplantation. Methodology. Analysis of a prospectively maintained database of patients treated with primary OCA transplantation from 2017 to 2021 with a minimum of one-year follow-up. Patient reported outcome measures (PROMs), complications, re-operations and failures were evaluated. Results. 37 patients with 37 knee OCA procedures were included (mean age 31.6 years [16–49 years]). Mean BMI 26.6 kg/m2 (19.1–35.9 kg/m2). The mean chondral defect size was 3cm2 (1.2–7.3 cm2). Mean duration of follow-up was 3.1 years (1–5.3 years). 16 patients underwent meniscal allograft transplantation (MAT), 6 underwent osteotomy and 4 underwent ligament reconstruction as concurrent procedures. Significant improvements in mean PROMs were noted at 12 months. 16 patients had reoperations of which 5 had more than one surgery. Of these patients 6 were related to OCA (mainly debridement and revision OCA in one patient), and the remainder were related to additional procedures including removal of plate in 2 patients. The overall failure rate was 1 in 37 patients (3%). Conclusions. Early experience of OCA as a treatment option for complex chondral and osteochondral lesions in the knee shows satisfactory results. The reoperation rate is high but at mean follow-up of 3.1 years the survival rate was 97%

The regenerative capacity of hyaline cartilage is greatly limited. To prevent the onset of osteoarthritis, cartilage defects have to be properly treated. Cartilage, tissue engineered by mean of bioactive glass (BG) scaffolds presents a promising approach. Until now, conventional BGs have been used mostly for bone regeneration, as they are able to form a hydroxyapatite (HA) layer and are therefore, less suited for cartilage reconstruction. The aim of this study is to compare two BGs based on a novel BG composition tailored specifically for cartilage (CAR12N) and patented by us with conventional BG (BG1393) with a similar topology. The highly porous scaffolds consisting of 100% BG (CAR12N, CAR12N with low Ca2+/Mg2+ and BG1393) were characterized and dynamically seeded with primary porcine articular chondrocytes (pACs) or primary human mesenchymal stem cells (hMSCs) for up to 21 days. Subsequently, cell viability, DNA and glycosaminoglycan contents, cartilage-specific gene and protein expression were evaluated. The manufacturing process led to a comparable high (over 80%) porosity in all scaffold variants. Ion release and pH profiles confirmed bioactivity for them. After both, 7 and 21 days, more than 60% of the total surfaces of all three glass scaffold variants was densely colonized by cells with a vitality rate of more than 80%. The GAG content was significantly higher in BG1393 colonized with pACs. In general, the GAG content was higher in pAC colonized scaffolds in comparison to those seeded with hMSCs. The gene expression of cartilage-specific collagen type II, aggrecan, SOX9 and FOXO1 could be detected in all scaffold variants, irrespectively whether seeded with pACs or hMSCs. Cartilage-specific ECM components could also be detected at the protein level. In conclusion, all three BGs allow the maintenance of the chondrogenic phenotype or chondrogenic differentiation of hMSCs and thus, they present a high potential for cartilage regeneration

Autologous osteochondral grafting has demonstrated positive outcomes for treating articular cartilage defects by replacing the damaged region with a cylindrical graft consisting of bone with a layer of cartilage, taken from a non-loadbearing region of the knee. Despite positive clinical use, factors that cause graft subsidence or poor integration are relatively unknown. The aim of this study was to develop finite element (FE) models of osteochondral grafts within a tibiofemoral joint and to investigate parameters affecting osteochondral graft stability. Initial experimental tests on cadaveric femurs were performed to calibrate the bone properties and graft-bone frictional forces for use in corresponding FE models, generated from µCT scan data. The effects of cartilage defects and osteochondral graft repair were measured by examining contact pressure changes using in vitro tests on a single cadaveric human tibiofemoral joint. Six defects were created in the femoral condyles which were subsequently treated with osteochondral autografts or metal pins. Matching µCT scan-based FE models were created, and the contact patches were compared. Sensitivity to graft bone properties was investigated. The bone material properties and graft-bone frictional forces were successfully calibrated from the initial tests with good resulting levels of agreement (CCC=0.87). The tibiofemoral joint experiment provided a range of cases to model. These cases were well captured experimentally and represented accurately in the FE models. Graft properties relative to host bone had large effects on immediate graft stability despite limited changes to resultant cartilage contact pressure. Model confidence was built through extensive validation and sensitivity testing, and demonstrated that specimen-specific properties were required to accurately represent graft behaviour. The results indicate that graft bone properties affect the immediate stability, which is important for the selection of allografts and design of future synthetic grafts. Acknowledgements. Supported by the EPSRC-EP/P001076

Mincing cartilage with commercially available shavers is increasingly used for treating focal cartilage defects. This study aimed to compare the impact of mincing bovine articular cartilage using different shaver blades on chondrocyte viability. Bovine articular cartilage was harvested using a scalpel or three different shaver blades (2.5 mm, 3.5 mm, or 4.2 mm) from a commercially available shaver. The cartilage obtained with a scalpel was minced into fragments smaller than 1 mm. 3. All four conditions were cultivated in a culture medium for seven days. After Day 1 and Day 7, metabolic activity, RNA isolation, and gene expression of anabolic (COL2A1, ACAN) and catabolic genes (MMP1, MMP13), Live/Dead staining and visualization using confocal microscopy, and flow cytometric characterization of minced cartilage chondrocytes were measured. The study found that mincing cartilage with shavers significantly reduced metabolic activity after one and seven days compared to scalpel mincing (p<0.001). Gene expression of anabolic genes was reduced, while catabolic genes were increased after day 7 in all shaver conditions. The MMP13/COL2A1 ratio was also increased in all shaver conditions. Confocal microscopy revealed a thin line of dead cells at the lesion site with viable cells below for the scalpel mincing and a higher number of dead cells diffusely distributed in the shaver conditions. After seven days, there was a significant decrease in viable cells in the shaver conditions compared to scalpel mincing (p<0.05). Flow cytometric characterization revealed fewer intact cells and proportionally more dead cells in all shaver conditions compared to the scalpel mincing. Mincing bovine articular cartilage with commercially available shavers reduces the viability of chondrocytes compared to scalpel mincing. This indicates that mincing cartilage with a shaver should be considered a matrix rather than a cell therapy. Further experimental and clinical studies are required to standardize the mincing process with a shaver. Acknowledgements: This study received unrestricted funding from KARL STORZ SE & Co. KG

The incidence of osteoarthritis (OA) is increasing in our younger population. OA development early in life is often related to cartilage damage, caused by (sport) injury or trauma. Detection of early knee OA is therefore crucial to target early treatment. However, early markers for OA prognosis or diagnosis are lacking. Hoffa's fat pad (HFP) is an emerging source for knee biomarkers, as it is easily accessible and shows important interaction with the homeostasis of the knee. In this study, we used Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) as a first approach. MALDI-MSI allows the study of tissue-specific molecular distributions. Therefore, we used MALDI-MSI to analyze the lipid profiles in the HFP of three patients with OA and three patients undergoing cartilage regenerative treatment. We demonstrate that the lipid profile of patients with OA is different from patients with cartilage defects. HFP of each patient were snap frozen directly after surgical resection and cryosectioned at 15 μm. Each slide was sublimed with Norharmane matrix and analyzed by MALDI-MSI in positive and negative ion modes at a lateral resolution of 50 μm on a RapifleX Tissue Typer. The difference between patient groups were analyzed using principle component analysis and linear discriminant analysis. Lipid identifications were obtained on an Orbitrap Elite™ Hybrid Ion Trap-Orbitrap Mass Spectrometer in data dependent acquisition mode and analyzed using Lipostar software. Linear discriminant analysis showed a specific lipid profile for each group (variance 33.94%). Score projections revealed a differential lipid spatial distribution of OA patients compared to cartilage defect patients. Among the lipids that differed significantly, for instance, the m/z 760.59 [M+H]. +. was associated to osteoarthritis and identified as glycerophospholipid (PC 34:1), a main component of biological membranes. Additionally, the samples were found to be intra-tissue heterogeneous, with molecular profiles found in adipose-, connective- and synovial tissue. These results suggest that lipid profiles in HFP could be useful for early OA detection. However, intra-tissue heterogeneity in HFP should be recognized when using HFP as a biomarker source

Damage to articular cartilage is difficult to treat, as it has a low capacity to regenerate. Biomimetic natural polymer scaffolds can potentially be used to regenerate cartilage. Collagen hyaluronic acid (CHyA) scaffolds have been developed in our laboratory to promote cell infiltration and repair of articular cartilage. However, the low mechanical properties of such scaffolds potentially limit their use to the treatment of small cartilage defects. 3D-printed polymers can provide a reinforcing framework in these scaffolds, thus allowing their application in the treatment of larger defects. The aim of this study was to create mechanically functional biomaterial scaffolds by incorporating a CHyA matrix into 3D-printed polymer meshes resulting in an integrated porous material composite with improved mechanical properties for repair of large cartilage defects. 3D-printed meshes were developed to facilitate an architecture suitable for nutrient flow, cell infiltration, and even CHyA incorporation. And the meshes were freeze dried in custom made moulds to create a pore structure suitable for chondrogenesis. Uniaxial compressive testing of the scaffolds revealed improved mechanical properties following reinforcement with printed meshes, with the compressive modulus increasing from 0.8kPa (alone) to 0.5MPa (reinforced structure). The reinforced scaffolds maintained interconnected pores with the mean pore diameter increasing from 130 to 175µm. The reinforcement had no negative impact on MSC viability, with 90.1% viability in reinforced scaffolds at day 7. The compressive modulus of the reinforced CHyA scaffold is close to native articular cartilage, suggesting that this approach can be used for treatment of large cartilage defects

Long-term regeneration of cartilage defects treated with tissue engineering constructs often fails because of insufficient integration with the host tissue. We hypothesize that construct integration will be improved when implants actively interact with and integrate into the subchondral bone. Growth and Differentiation Factor 5 (GDF-5) is known to support maturation of chondrocytes and to enhance chondrogenic differentiation and hypertrophy of mesenchymal stromal cells (MSC). Therefore, we investigated whether GDF-5 is capable to stimulate endochondral ossification of MSC in vitro and in vivo and would, thus, be a promising candidate for augmenting fibrin glue in order to support integration of tissue engineering constructs into the subchondral bone plate. To evaluate the adhesive strength of fibrin glue versus BioGlue. ®. , a commercially available glue used in vascular surgery, an ex vivo cadaver study was performed and adhesion strength was measured via pull-out testing. MSC were suspended in fibrin glue and cultivated in chondrogenic medium with and without 150 ng/mL GDF-5. After 4 weeks, the formed cartilage was evaluated and half of the constructs were implanted subcutaneously into immunodeficient mice. Endochondral ossification was evaluated after 2 and 4 weeks histologically and by microCT analysis. BioGlue. ®. and GDF-5-augmented fibrin glue were tested for 4 weeks in a minipig cartilage defect model to assess their orthotopic biocompatibility. Pull-out testing revealed sufficient adhesive strength of fibrin glue to fix polymeric CellCoTec constructs in 6 mm cartilage defects, however, BioGlue. ®. showed significantly higher adhesive power. In vitro chondrogenesis of MSC under GDF-5 treatment resulted in equal GAG deposition and COLIIa1 and ACAN gene expression compared to controls. Importantly, significantly increased ALP-activity under treatment with GDF-5 on day 28 indicated enhanced hypertrophic differentiation compared to controls. In vivo, MSC-fibrin constructs pre-cultured with GDF-5 developed a significantly higher bone volume on day 14 and 28 compared to controls. When pre-cultured with GDF-5 constructs showed furthermore a significantly higher bone compactness (bone surface/bone volume coefficient) than controls, and thus revealed a higher maturity of the formed bone at 2 weeks and 4 weeks. Orthotopic biocompatibility testing in minipigs showed good defect filling and no adverse reactions of the subchondral bone plate for defects treated with GDF-5-augmented fibrin glue. Defects treated with BioGlue. ®. , however, showed considerable subchondral bone lysis. Thus, BioGlue. ®. – despite its adhesive strength – should not be used for construct fixation in cartilage defects. GDF-5-augmented fibrin glue is considered promising, because of a combination of the adhesive strength of fibrin with an enhanced osteochondral activity of GDF-5 on MSC. Next step is to perform a large animal study to unravel whether GDF-5 stimulated endochondral ossification can improve scaffold integration in an orthotopic cartilage defect model