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Cartilage is an avascular and aneural connective tissue with poor self-healing capability. Recently, tissue engineering opens up new horizons for staving off or treating cartilage lesions. In this work, kartogenin (KGN), a small chondro-inductive molecule, was loaded into poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs), which in turn was embedded in a silk fibroin (SF) scaffold, to prepare an appropriate microenvironment for mesenchymal stem cell (MSC) differentiation. In this research, SF was opted to serve as a scaffold based on its approved biocompatibility and non-toxicity, excellent mechanical properties and processability. The data obtained from this study show that entrapment of KGN in NPs provides sustained release, which could promote the differentiation of MSCs into chondrocytes. Likewise, the scaffold containing KGN-loaded NPs induces glycosaminoglycan production by the seeded MSCs. The introduction of NPs into the scaffold, meanwhile, elevated the compressive strength of the structures (more than two times) without any significant effect on their swelling behavior. Taken together, the authors’ findings demonstrate that the prepared scaffold, with an optimal structure, could be a potential candidate for cartilage tissue regeneration.

Pendahuluan: Osteoarthritis merupakan penyakit degeneratif sendi paling umum terjadi dan telah menyerang lebih jutaan orang di seluruh dunia. Walaupun angka prevalensi dan mortalitas dari penyakit ini terus meningkat setiap tahunnya, tetapi sampai saat ini belum ada terapi yang mampu menghambat atau bahkan menghentikan perjalanan penyakit dari osteoarthritis. Baru-baru ini, sebuah molekul kecil bernama kartogenin (KGN) telah dilaporkan mampu melakukan perbaikan dan peningkatan kualitas dari kartilago sendi dan tulang subkondral pada hewan yang menderita osteoarthritis. Selanjutnya, untuk dapat menghantarkan molekul tersebut secara efektif dan efisien para penelitian kemudian juga menggembangkan sebuah sistem penghantaran obat yang baru bernama nanocrystal- polymer particles (NPP). Penghantaran KGN dengan NPP telah dilaporkan tidak hanya mampu meningkatkan efektifitas dari molekul tersebut, tetapi juga menurunkan kemungkinan efek samping yang timbul dari terapi tersebut. Pembahasan: Studi literatur ini menunjukkan bahwa KGN-NPP terbukti mampu menginduksi diferensiasi mesenchymal stem cells (MSCs) menjadi kondrosit yang ditandai dengan meningkatnya ekspresi dari kolagen tipe 2 (Col2) dan Aggrecan hingga 5.5 kali lipat. Selain itu, kerusakan pada kartilago sendi juga telah terbukti dapat dicegah hingga 100% pada studi in vivo dengan ditemukannya skor OARSI yang mendekati angka nol. Bukan hanya itu saja, KGN-NPP juga telah terbukti mampu meningkatkan volume dari tulang subkondral. Hal tersebut merupakan bukti bahwa KGN-NPP tidak hanya mampu bertindak sebagai kondroprotektif, tetapi juga mampu meregenerasi kerusakan yang telah terjadi sebelumnya. Kesimpulan: Dengan segala keunggulan yang dimiliki, dapat disimpulkan bahwa KGN-NPP memiliki potensi yang menjanjikan untuk menjadi terapi yang efektif bagi semua penderita osteoarthritis di seluruh dunia. Kata Kunci: kartogenin, nanocrystals-polymer particle, osteoarthritis

Background Regeneration of articular cartilage poses a tremendous challenge due to its limited self-repair capability and inflammation at the damaged site. To generate the desired structures that mimic the structure of native tissue, microtissues with repeated functional units such as cell aggregates have been developed. Multicellular aggregates of mesenchymal stem cells (MSCs) can be used as microscale building blocks of cartilage due to their potential for cell-cell contact, cell proliferation, and differentiation. Methods Chondrogenic microtissues were developed through incorporation of kartogenin-releasing poly (lactic-co-glycolic acid) (PLGA) microparticles (KGN-MP) within the MSC aggregates. The chondrogenic potential of KGN-MP treated MSC aggregates was proven in vitro by studying the chondrogenic markers at the RNA level and histological analysis. In order to address the inflammatory responses at the defect site, the microtissues were delivered in vivo via an injectable, anti-inflammatory hydrogel that contained gelatin methacryloyl (GelMA) loaded with curcumin (Cur). Results The KGN-MPs were fabricated to support MSCs during cartilage differentiation. According to real-time RT-PCR analysis, the presence of KGN in the aggregates led to the expression of cartilage markers by the MSCs. Both toluidine blue (TB) and safranin O (SO) staining demonstrated homogeneous glycosaminoglycan production throughout the KGN-MP incorporated MSC aggregates. The curcumin treatment efficiently reduced the expressions of hypertrophy markers by MSCs in vitro. The in vivo results showed that implantation of chondrogenic microtissues (KGN-MP incorporated MSC aggregates) using the curcumin loaded GelMA hydrogel resulted in cartilage tissue regeneration that had characteristic features close to the natural hyaline cartilage according to observational and histological results. Conclusions The use of this novel construct that contained chondrogenic cell blocks and curcumin is highly desired for cartilage regeneration. Graphical abstract

Objective To explore the effect of kartogenin (KGN) on proliferation and chondrogenic differentiation of human umbilical cord mesenchymal stem cells (hUCMSC) in vitro, and the synergistic effects of KGN and transforming growth factor (TGF)‐β3 on hUCMSC. Methods Human umbilical cord mesenchymal stem cells were isolated and cultured. Then the differentiation properties were identified by flow cytometry analysis. HUCMSC were divided into four groups: control group, KGN group, TGF‐β3 group, and TK group (with TGF‐β3 and KGN added into the medium simultaneously). Cells in all groups were induced for 21 days using the suspension ball culture method. Hematoxylin and eosin, immunofluorescence, and Alcian blue staining were used to analyze chondrogenic differentiation. Real‐time reverse transcriptase polymerase chain reaction was performed to investigate genes associated with chondrogenic differentiation. Result Hematoxylin and eosin staining showed that cells in the TGF‐β3 group and the TK group had formed cartilage‐like tissue after 21 days of culture. The results of immunofluorescence and Alcian blue staining showed that compared with the control group, cells in the KGN and TGF‐β3 groups demonstrated increased secretion of aggrecan after 21 days of culture. In addition, cells in the group combining KGN with TGF‐β3 (5.587 ± 0.27, P

Ultrasound-responsive microspheres (MPs) derived from natural polysaccharides and injectable hydrogels have been widely investigated as a biocompatible, biodegradable, and controllable drug delivery system and cell scaffolds for tissue engineering. In this study, kartogenin (KGN) loaded poly (lactide-co-glycolic acid) (PLGA) MPs (MPs@KGN) were fabricated by premix membrane emulsification (PME) method which were sonicated by an ultrasound transducer. Furthermore, carboxymethyl chitosan-oxidized chondroitin sulfate (CMC-OCS) hydrogel were prepared via the Schiff’ base reaction-embedded MPs to produce a CMC-OCS/MPs scaffold. In the current work, morphology, mechanical property, porosity determination, swelling property, in vitro degradation, KGN release from scaffolds, cytotoxicity, and cell bioactivity were investigated. The results showed that MPs presented an obvious collapse after ultrasound treatment. The embedded PLGA MPs could enhance the compressive elastic modulus of soft CMC-OCS hydrogel. The cumulative release KGN from MPs exhibited a slow rate which would display an appropriate collapse after ultrasound, allowing KGN to maintain a continuous concentration for at least 28 days. Moreover, the composite CMC-OCS@MPs scaffolds exhibited faster gelation, lower swelling ratio, and lower in vitro degradation. CCK-8 and LIVE/DEAD staining showed these scaffolds did not influence rabbit bone marrow mesenchymal stem cells (rBMMSCs) proliferation. Then these scaffolds were cultured with rBMMSCs for 2 weeks, and the immunofluorescent staining of collagen II (COL-2) showed that CMC-OCS hydrogel embedded with MPs@KGN (CMC-OCS@MPs@KGN) with ultrasound had the ability to increase the COL-2 synthesis. Overall, due to the improved mechanical property and the ability of sustained KGN release, this injectable hydrogel with ultrasound-responsive property is a promising system for cartilage tissue engineering.

Rationale The small molecule Kartogenin (KGN) promotes cartilage regeneration in osteoarthritis (OA) by activating stem cells differentiation, but its pharmacological mode-of-action remains unclear. KGN can be cleaved into 4-aminobiphenyl (4-ABP) and phthalic acid (PA) following enzymolysis of an amide bond. Therefore, this study investigated whether 4-ABP or PA exerted the same action as KGN. Methods KGN, 4-ABP and PA were analyzed in cartilage of mice after oral, intravenous or intra-articular administration of KGN by liquid chromatography-mass spectrometry method. Their effect on proliferation and chondrogenic differentiation of mesenchymal stem cells (MSC) was evaluated in vitro. Furthermore, their effect on cartilage preservation was tested in mice OA model induced by destabilization of medial meniscus. OA severity was quantified using OARSI histological scoring. Transcriptional analysis was used to find the possible targets of the chemicals, which were further validated. Results We demonstrated that while oral or intra-articular KGN delivery effectively ameliorated OA phenotypes in mice, only 4-ABP was detectable in cartilage. 4-ABP could induce chondrogenic differentiation and proliferation of MSC in vitro and promote cartilage repair in OA mouse models mainly by increasing the number of CD44+/CD105+ stem-cell and prevention of matrix loss. These effect of 4-ABP was stronger than that of KGN. Transcriptional profiling of 4-ABP-stimulated MSC suggested that RPS6KA2 and the PI3K-Akt pathway were 4-ABP targets; 4-ABP could activate the PI3K-Akt pathway to promote MSC proliferation and repair OA injury, which was blocked in RPS6KA2-knockdown MSC or RPS6KA2-deficient mice. Conclusion 4-ABP bio-distribution in cartilage promotes proliferation and chondrogenic differentiation of MSC, and repairs osteoarthritic lesions via PI3K-Akt pathway activation.

Background The meniscus tear is one of the most common knee injuries particularly seen in athletes and aging populations. Subchondral bone sclerosis, irreparable joint damage, and the early onset of osteoarthritis make the injured meniscus heal difficultly. Methods The study was performed by in vitro and in vivo experiments. The in vitro experiments were carried out using the bone marrow stem cells (BMSCs) isolated from the rabbits, and the stemness of the BMSCs was tested by immunostaining. The BMSCs positively expressed stem cell markers were cultured with various concentrations of kartogenin (KGN) for 2 weeks. The chondrogenesis of BMSCs induced by KGN was examined by histochemical staining and quantitative RT-PCR. The in vivo experiments were completed by a rabbit model. Three holes were created in each meniscus by a biopsy punch. The rabbits were treated with four different conditions in each group. Group 1 was treated with 20 μl of saline (saline); group 2 was treated with 5 μl of 100 μM KGN and 15 μl saline (KGN); group 3 was treated with 5 μl of 100 μM KGN, 5 μl of 10,000 U/ ml thrombin, and 10 μl of PRP (KGN+PRP); group 4 was treated with 10,000 BMSCs in 10 μl of PRP, 5 μl of saline solution, and 5 μl of 10,000 U/ml thrombin (PRP+BMSC); group 5 was treated with 10,000 BMSCs in 10 μl of PRP, 5 μl of 100 μM KGN, and 5 μl of 10,000 U/ml thrombin (KGN+PRP+BMSC). The menisci were collected at day 90 post-surgery for gross inspection and histochemical analysis. Results The histochemical staining showed that KGN induced chondrogenesis of BMSCs in a concentration-dependent manner. The RT-PCR results indicated that chondrocyte-related genes were also increased in the BMSCs cultured with KGN in a dose-dependent manner. The in vivo results showed that large unhealed wound areas were still found in the wounds treated with saline and KGN groups. The wounds treated with BMSCs-containing PRP gel healed much faster than the wounds treated without BMSCs. Furthermore, the wounds treated with BMSCs-containing KGN-PRP gel have healed completely and formed more cartilage-like tissues than the wounds treated with BMSCs-containing PRP gel. Conclusions BMSCs could be differentiated into chondrocytes when they were cultured with KGN-PRP gel in vitro and formed more cartilage-like tissues in the wounded rabbit meniscus when the wounds were treated with BMSCs-containing KGN-PRP gel. The results indicated that the BMSCs-containing KGN-PRP gel is a good substitute for injured meniscus repair and regeneration.

A dual-layer biomimetic cartilage scaffold was prepared by mimicking the structural design, chemical cues and mechanical characteristics of mature articular cartilage. The surface layer was made from collagen (COL), chitosan (CS) and hyaluronic acid sodium (HAS). The transitional layer with microtubule array structure was prepared with COL, CS and silk fibroin (SF). The PLAG microspheres containing kartogenin (KGN) and the polylysine-heparin sodium nanoparticles containing TGF-β1 (TPHNs) were constructed for the surface, transitional layer, respectively. The SEM result showed that the dual-layer composite scaffold had a double structure similar to natural cartilage. The vitro biocompatibility experiment showed that the biomimetic cartilage scaffold with orientated porous structure was more conducive to the proliferation and adhesion of BMSCs. A rabbit KOA cartilage defect model was established and biomimetic cartilage scaffolds were implanted in the defect area. Compared with the surface layer and transitional layer scaffolds group, the results of dual-layer biomimetic cartilage scaffold group showed that the defects had been completely filled, the boundary between new cartilage and surrounding tissue was difficult to identify, and the morphology of cells in repair tissue was almost in accordance with the normal cartilage after 16 weeks. All those results indicated that the biomimetic cartilage scaffold could effectively repair the defect of KOA, which is related to the fact that the scaffold could guide the morphology, orientation, and proliferation and differentiation of BMSCs. This work could potentially lead to the development of multilayer scaffolds mimicking the zonal organization of articular cartilage.

Repair of articular cartilage defects is a challenging aspect of clinical treatment. Kartogenin (KGN), a small molecular compound, can induce the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into chondrocytes. Here, we constructed a scaffold based on chondrocyte extracellular matrix (CECM) and poly(lactic-co-glycolic acid) (PLGA) microspheres (MP), which can slowly release KGN, thus enhancing its efficiency. Cell adhesion, live/dead staining, and CCK-8 results indicated that the PLGA(KGN)/CECM scaffold exhibited good biocompatibility. Histological staining and quantitative analysis demonstrated the ability of the PLGA(KGN)/CECM composite scaffold to promote the differentiation of BMSCs. Macroscopic observations, histological tests, and specific marker analysis showed that the regenerated tissues possessed characteristics similar to those of normal hyaline cartilage in a rabbit model. Use of the PLGA(KGN)/CECM scaffold may mimic the regenerative microenvironment, thereby promoting chondrogenic differentiation of BMSCs in vitro and in vivo. Therefore, this innovative composite scaffold may represent a promising approach for acellular cartilage tissue engineering.

Background: Treatment of cartilage lesions is clinically challenging. A previous study demonstrated that a hyaluronic acid hydrogel ( m-HA) with kartogenin (KGN)-loaded PLGA nanoparticles ( m-HA+KGN treatment) achieved superior cartilage repair in a rabbit model. However, large animals serve as a bridge to translate animal outcomes into the clinic. Hypotheses: (1) m-HA+KGN treatment could facilitate hyaline cartilage and subchondral bone tissue repair in a porcine model. (2) Defect size and type (full-thickness chondral vs osteochondral) influence the therapeutic efficacy of m-HA+KGN treatment. Study Design: Controlled laboratory study. Methods: 48 minipigs were randomized into 3 treatment groups: m-HA hydrogel with KGN-loaded PLGA nanoparticles ( m-HA+KGN treatment), m-HA hydrogel ( m-HA treatment), and untreated (blank treatment). Full-thickness chondral (6.5 mm or 8.5 mm in diameter) or osteochondral (6.5 mm or 8.5 mm in diameter; 5-mm depth) defects were prepared in the medial femoral condyle. At 6 and 12 months postoperatively, defect repair was assessed by macroscopic appearance, magnetic resonance imaging (MRI), micro–computed tomography (µCT), and histologic and biomechanical tests. Results: The m-HA+KGN group exhibited superior gross and histological healing after evaluation at 6 and 12 months postoperatively. Improved quality of the repaired cartilage demonstrated by MRI and better subchondral bone reconstruction assessed by µCT were observed in the m-HA+KGN group. The m-HA+KGN group showed more hyaline-like cartilage exhibited by histological staining in terms of extracellular matrix, cartilage lacuna, and type II collagen. The biomechanical properties were improved in the m-HA+KGN group. With m-HA+KGN treatment, defects with a diameter of 6.5 mm or full-thickness chondral-type defects possessed significantly higher ICRS macroscopic and histological scores compared with diameter 8.5 mm or osteochondral-type defects. Conclusion: (1) m-HA+KGN treatment facilitated hyaline cartilage and subchondral bone tissue repair in a porcine model at the 12-month follow-up. (2) m-HA+KGN treatment demonstrated better therapeutic efficacy in defects with a diameter of 6.5 mm or full-thickness chondral-type defects. Clinical Relevance: This study verified the efficacy of this innovative KGN release system on cartilage repair. The KGN release system can be injected into defect sites arthroscopically. This convenient and minimally invasive operation holds important prospects for clinical application.

Objective Human mesenchymal stem cells (hMSCs) are a promising source for regenerative medicine, especially mesodermal lineages. Clinical applications require an understanding of the mechanisms for transcriptional control to maintain the desired cell type. The aim of this study was to identify novel markers for differentiation of hMSCs into bone or cartilage with the use of Kartogenin, by RNA analysis using microarray technology, and explore the role of RhoA-Rho associated protein kinase (ROCK) inhibition in these. Methods Commercial human bone marrow derived primary mesenchymal stem cells were purchased from ATCC. Cells were differentiated in vitro in 2-dimensional cultures using Kartogenin as the main cartilage inducer and bone morphogenetic protein 2 for bone differentiation; cells were cultured with and without ROCK inhibitor Y-27632. After 21 days of culture, whole RNA was extracted and analyzed via Affimetrix microarrays. The most significant hits were validated by quantitative polymerase chain reaction. Results We found a total of 1,757 genes that were either up- or downregulated on differentiation, when compared to P1 hMSC (control) at day 0 of differentiation. Two members of the Serpin superfamily, SERPINA9 and SERPINB2, were significantly upregulated in the cartilage groups, whereas they were unchanged in the bone groups with and without ROCK inhibition. Conclusions SERPINA9 and SERPINB2 are novel differentiation markers, and molecular regulator candidates for hMSC lineage commitment toward bone and cartilage, providing a new tool for regenerative medicine. Our study highlights the roles of these 2 genes, with significant upregulation of both in cell cultures stimulated with Kartogenin.

Osteoarthritis (OA) is the most common form of joint disease and a leading cause of physical disability, there is an urgent need to attenuate the progression of OA. Intra-articular (IA) injection is an effective treatment for joints diseases, however, the therapeutic effects mostly depend on the efficacy of drug duration in joints. Drug delivery system can provide drug-controlled release and reduce the number of IA injection. In this study, amphiphilic polyurethanes with pendant amino group were synthesized and amide bonds were formed between the amine group of polyurethane and the carboxyl group of kartogenin (KGN), a small molecular reported to show both regenerative and protective effects on cartilage. Our results showed that KGN-conjugated polyurethane nanoparticles (PN-KGN) were spherical and regular in shape with an average size of 25 nm and could sustained and controlled release of KGN in vitro. PN-KGN showed no cytotoxicity and pro-inflammatory effects on chondrocytes. The therapeutic effects in OA model showed that IA injection of KGN could attenuate the progress of OA, however, the cartilage degeneration became obviously at 12 weeks with matrix loss and vertical fissures. By contrast, IA injection of PN-KGN showed less cartilage degeneration with significant lower OARSI scores even at 12 weeks, indicating PN-KGN could further arrest the development of OA. Immunohistochemistry also validated that IA injection of PN-KGN retained the normal compositions of cartilage matrix, with much stronger Col II staining and less Col I staining. In conclusion, IA injection of PN-KGN is a better potential strategy to treat OA, with long-time cartilage protection and less IA injections.

Objectives After an injury, the biological reattachment of tendon to bone is a challenge because healing takes place between a soft (tendon) and a hard (bone) tissue. Even after healing, the transition zone in the enthesis is not completely regenerated, making it susceptible to re-injury. In this study, we aimed to regenerate Achilles tendon entheses (ATEs) in wounded rats using a combination of kartogenin (KGN) and platelet-rich plasma (PRP). Methods Wounds created in rat ATEs were given three different treatments: kartogenin platelet-rich plasma (KGN-PRP); PRP; or saline (control), followed by histological and immunochemical analyses, and mechanical testing of the rat ATEs after three months of healing. Results Histological analysis showed well organised arrangement of collagen fibres and proteoglycan formation in the wounded ATEs in the KGN-PRP group. Furthermore, immunohistochemical analysis revealed fibrocartilage formation in the KGN-PRP-treated ATEs, evidenced by the presence of both collagen I and II in the healed ATE. Larger positively stained collagen III areas were found in both PRP and saline groups than those in the KGN-PRP group. Chondrocyte-related genes, SOX9 and collagen II, and tenocyte-related genes, collagen I and scleraxis (SCX), were also upregulated by KGN-PRP. Moreover, mechanical testing results showed higher ultimate tensile strength in the KGN-PRP group than in the saline control group. In contrast, PRP treatment appeared to have healed the injured ATE but induced no apparent formation of fibrocartilage. The saline-treated group showed poor healing without fibrocartilage tissue formation in the ATEs. Conclusions Our results show that injection of KGN-PRP induces fibrocartilage formation in the wounded rat ATEs. Hence, KGN-PRP may be a clinically relevant, biological approach to regenerate injured enthesis effectively. Cite this article: J. Zhang, T. Yuan, N. Zheng, Y. Zhou, M. V. Hogan, J. H-C. Wang. The combined use of kartogenin and platelet-rich plasma promotes fibrocartilage formation in the wounded rat Achilles tendon entheses. Bone Joint Res 2017;6:231–244. DOI: 10.1302/2046-3758.64.BJR-2017-0268.R1.

Treatment of tendon-bone junction injuries is a challenge because tendon-bone interface often heals poorly and the fibrocartilage zone, which reduces stress concentration, at the interface is not formed. In this study, we used a compound called kartogenin (KGN) with platelet-rich plasma (PRP) to induce the formation of fibrocartilage zone in a rat tendon graft-bone tunnel model. The experimental rats received KGN-PRP or PRP injections in the tendon graft-bone tunnel interface. The control group received saline. After 4, 8 and 12 weeks, Safranin O staining of the tendon graft-bone tunnels revealed abundant proteoglycans in the KGN-PRP group indicating the formation of cartilage-like transition zone. Immunohistochemical and immuno-fluorescence staining revealed collagen types I (Col-I) and II (Col-II) in the newly formed fibrocartilage zone. Both fibrocartilage zone formation and maturation were healing time dependent. In contrast, the PRP and saline control groups had no cartilage-like tissues and minimal Col-I and Col-II staining. Some gaps were also present in the saline control group. Finally, pull-out strength in the KGN-PRP-treated group at 8 weeks was 1.4-fold higher than the PRP-treated group and 1.6-fold higher than the saline control group. These findings indicate that KGN, with PRP as a carrier, promotes the formation of fibrocartilage zone between the tendon graft and bone interface. Thus, KGN-PRP may be used as a convenient cell-free therapy in clinics to promote fibrocartilage zone formation in rotator calf repair and anterior cruciate ligament reconstruction, thereby enhancing the mechanical strength of the tendon-bone interface and hence the clinical outcome of these procedures. Copyright © 2017 John Wiley & Sons, Ltd.

To provide a vehicle for sustained release of cartilage-protective agent for the potential application of osteoarthritis (OA) treatment, we developed a kartogenin (KGN)-incorporated thermogel for intra-articular injection. We fabricated a poly(lactide-co-glycolide)-block-poly(ethylene glycol)-block-poly(lactide-co-glycolide) (PLGA–PEG–PLGA) thermogel as a KGN carrier for IA injection. OA chondrocytes were cultured in thermogel with or with no KGN to investigate the effect of KGN thermogel on cartilage matrix. The in vivo effect of KGN thermogel on OA was examined in a rabbit OA model. The KGN thermogel showed a sustained in vitro release of KGN for 3 weeks. OA chondrocytes proliferated well both in thermogel and KGN thermogel. In addition, OA chondrocytes produced higher amount of [type 2 collagen (COL-2) and glycosaminoglycan (GAG)], as well as lower level of matrix metalloproteinase 13 (MMP-13) in KGN thermogel that those in thermogel with no addition of KGN. The gene analysis supported that KGN thermogel enhanced expression of hyaline-cartilage specific genes Col 2 and AGC, and inhibited the expression of MMP-13. Compared with intra-articular injection of saline or thermogel containing no KGN, KGN thermogel can enhance cartilage regeneration and inhibit joint inflammation of arthritic knees in a rabbit ACLT-induced OA model at 3 weeks after the injection. Therefore, the KGN-incorporated PLGA–PEG–PLGA thermogel may provide a novel treatment modality for OA treatment with IA injection.

Objective To measure the inductive effect of kartogenin on matrix metalloproteinase-2 levels during the differentiation of human bone marrow mesenchymal stem cells (hMSCs) into chondrocytes in vitro. Methods In vitro cultured bone marrow hMSCs were grown to the logarithmic phase and then divided into three groups: control group (0 µM kartogenin), 1 µM kartogenin group and 10 µM kartogenin group. After 72 h of culture, cell proliferation and differentiation were observed microscopically. Matrix metalloproteinase-2 (MMP-2) in the cell supernatant and type II collagen levels in the cells were detected by enzyme linked immunosorbent assay and immunofluorescence staining, respectively. Results Kartogenin induced the proliferation and differentiation of hMSCs. With the increase of kartogenin concentration, the level of type II collagen was increased, while the level of MMP-2 decreased. Conclusion These findings indicate that kartogenin can induce hMSCs to differentiate into chondrocytes, and with the increase of kartogenin concentration, degeneration of the cartilage extracellular matrix may be inhibited.

Cartilage defect repair remains a great clinical challenge due to the limited self-regeneration capacity of cartilage tissue. Surgical treatment of injured cartilage is rather difficult due to the narrow space in the articular cavity and irregular defect area. Herein, we designed and fabricated chondrogenic and physiological-temperature-triggered shape-memory ternary scaffolds for cell-free cartilage repair, where the poly (glycerol sebacate) (PGS) networks ensured elasticity and shape recovery, crystallized poly (1,3-propylene sebacate) (PPS) acted as switchable phase, and immobilized bioactive kartogenin (KGN) endowed the scaffolds with chondrogenic capacity. The resultant scaffolds exhibited shape-memory properties with shape-memory fixed ratio of 98% and recovered ratio of 97% at 37°C for PPS/PGS/KGN-100, indicating a good potential for minimally invasive implantation. The scaffolds gradually degraded in Dulbecco's phosphate-buffered saline and released KGN up to 12 weeks in vitro. In addition, the scaffolds promoted chondrogenic differentiation while inhibiting osteogenic differentiation of bone marrow-derived mesenchymal stem cells in a concentration-dependent manner and cartilage regeneration in full-thickness defects of rat femoropatellar groove for 12 weeks. Consequently, the PPS/PGS/KGN-100 scaffolds stimulated the formation of an overlying layer of neocartilage mimicking the characteristic architecture of native articular cartilage even in the absence of exogenous growth factors and seeded cells. This study provides much inspiration for future research on cartilage tissue engineering. STATEMENT OF SIGNIFICANCE: There are two crucial challenges for cartilage defect repair: the lack of self-regeneration capacity of cartilage tissue and difficult scaffold implantation via traditional open surgery due to space-limited joints. Herein, bioactive body-temperature-responsive shape memory scaffolds are designed to simultaneously address the challenges. The scaffolds can be readily implanted by minimally invasive approach and recover by body-temperature of patient. The integration of kartogenin endows scaffolds the bioactivity, leading to the first example of bulk shape-memory scaffolds for cell-free cartilage repair. These characteristics make the scaffolds advantageous for clinical translation. Moreover, our developed material is easy to be functionalized due to the presence of extensive free hydroxyl groups and provides a versatile platform to design diverse functional shape memory biomaterials.

Human cartilage has relatively slow metabolism compared to other normal tissues. Cartilage damage is of great clinical consequence since cartilage has limited intrinsic healing potential. Cartilage tissue engineering is a rapidly emerging field that holds great promise for tissue function repair and artificial/engineered tissue substitutes. However, current clinical therapies for cartilage repair are less than satisfactory and rarely recover full function or return the diseased tissue to its native healthy state. Kartogenin (KGN), a small molecule, can promote chondrocyte differentiation both in vitro and in vivo. The purpose of this research is to optimize the chondrogenic process in mesenchymal stem cell (MSC)-based chondrogenic constructs with KGN for potential use in cartilage tissue engineering. In this study, we demonstrate that KGN treatment can promote MSC condensation and cell cluster formation within a tri-copolymer scaffold. Expression of Acan, Sox9, and Col2a1 was significantly up-regulated in three-dimensional (3D) culture conditions. The lacuna-like structure showed active deposition of type II collagen and aggrecan deposition. We expect these results will open new avenues for the use of small molecules in chondrogenic differentiation protocols in combination with scaffolds, which may yield better strategies for cartilage tissue engineering.

Resumo O objetivo deste estudo foi avaliar o reparo da cartilagem hialina equina, por meio de análises macroscópica (através de videoartroscopia) e histológica (através de fragmentos de biopsia), em defeitos condrais induzidos na tróclea lateral do fêmur tratados pela técnica de microperfurações subcondral associada ou não com administração intra-articular de cartogenina. Foram utilizados seis equinos pesando em média (±DP) 342±1,58 kg, com a idade aproximada de 7,2±1,30 anos e escore corporal de 7,1±0,75, que foram submetidos a videoartroscopia para indução da lesão condral de 1 cm2 na tróclea lateral do fêmur e realização da técnica de microperfuração do osso subcondral de ambos os joelhos. Foram realizadas quatro aplicações semanais com 20 μM de cartogenina intra-articulares em um dos joelhos (grupo tratado) e solução de ringer com lactato na articulação contralateral (grupo controle). Após o período de 60 dias, foram feitas as avaliações macroscópicas, através de videoartroscopias, e histológicas, através de biopsia. Não foram observadas diferenças significativas nos escores macroscópicos e histológicos para reparação condral entre animais dos grupos tratados e não tratados (P>0,05). De modo geral, a porcentagem média de cartilagem hialina no tecido de reparo (17,5%) foi condizente com a literatura internacional usando outros tipos de perfuração condral. Entretanto, não se observaram diferenças estatísticas entre grupos (P>0,05). A terapia com cartogenina, segundo protocolo utilizado, não produziu melhora do processo cicatricial em lesões condrais induzidas e tratadas com microperfurações na tróclea lateral do fêmur em equinos. Abstract The aim of this study was to evaluate the joint cartilage repair by macroscopic (via arthroscopy) and histological (biopsy fragments) analyses in chondral defects induced into equine femoral trochlea treated by microperforation associated with or without intra-articular administration of kartogenin. Six horses weighing 342±1.58 kg (mean ± SD), aged approximately 7.2±1.30 years and with a body condition score of 7.1±0.75, were used. The horses underwent arthroscopy for induction of 1-cm2 chondral lesions in lateral femoral trochlea immediately treated by microperforation of the subchondral bone of both knees. Four weekly intra-articular injections of kartogenin (20μM) in one knee (treated group) and Ringer lactate solution in the contralateral joint (control group) were performed during the postoperative period. After 60 days, macroscopic evaluations were performed by video-arthroscopy, and biopsy samples of the repair tissue were taken for histopathological healing evaluation. No significant change was observed in macroscopic and histological scores for chondral healing between treated and untreated groups (P>0.05). The overall mean percentage of hyaline cartilage in both groups (17.5%) was consistent with other international studies using other types of chondral microperforation; however, no statistical differences were observed between groups (P>0.05). In conclusion, the therapy with kartogenin, according to the used protocol, did not produce any macroscopic and histological healing improvement in induced chondral lesions treated with microperforations in equine femoral trochlea.

Entheses are the insertion sites where tendons and ligaments attach to bone. Commonly, entheses are subject to overuse injuries, and tendon-to-bone healing is poor because the healing has occurred between 2 different tissues: hard tissue (bone) and soft tissue (tendon). It is necessary to form the zonal arrangement of the enthesis region in vivo after repair.

An efficient treatment for osteoarthritis (OA) can benefit from the local release of a high therapeutic dose over an extended period of time. Such a treatment will minimize systemic side effects and avoid the inconvenience of frequent injections. To this aim, nanocrystal-polymer particles (NPPs) are developed by combining the advantages of nanotechnology and microparticles. Nanocrystals are produced by wet milling kartogenin (KGN), which is known to promote chondrogenesis and to foster chondroprotection. A fluorescent biodegradable polymer is synthesized for intravital particle tracking. Polymer microparticles with 320 nm embedded KGN nanocrystals (KGN-NPPs) show a high drug loading of 31.5% (w/w) and an extended drug release of 62% over 3 months. In vitro, these particles do not alter mitochondrial activity in cultured human OA synoviocytes. In vivo, KGN-NPPs demonstrate higher bioactivity than a KGN solution in a murine mechanistic OA model based on histological assessment (Osteoarthritis Research Society International score), epiphyseal thickness (microcomputed tomography), OA biomarkers (e.g., vascular endothelial growth factor, Adamts5), and prolonged intra-articular persistence (fluorescence analysis). This work provides proof-of-concept of a novel and innovative extended drug delivery system with the potential to treat human OA.

Tracheal stenosis is one of major challenging issues in clinical medicine because of the poor intrinsic ability of tracheal cartilage for repair. Tissue engineering provides an alternative method for the treatment of tracheal defects by generating replacement tracheal structures. In this study, we fabricated coaxial electrospun fibers using poly(L-lactic acid-co-caprolactone) and collagen solution as shell fluid and kartogenin solution as core fluid. Scanning electron microscope and transmission electron microscope images demonstrated that nanofibers had uniform and smooth structure. The kartogenin released from the scaffolds in a sustained and stable manner for about 2 months. The bioactivity of released kartogenin was evaluated by its effect on maintain the synthesis of type II collagen and glycosaminoglycans by chondrocytes. The proliferation and morphology analyses of mesenchymal stems cells derived from bone marrow of rabbits indicated the good biocompatibility of the fabricated nanofibrous scaffold. Meanwhile, the chondrogenic differentiation of bone marrow mesenchymal stem cells cultured on core-shell nanofibrous scaffold was evaluated by real-time polymerase chain reaction. The results suggested that the core-shell nanofibrous scaffold with kartogenin could promote the chondrogenic differentiation ability of bone marrow mesenchymal stem cells. Overall, the core-shell nanofibrous scaffold could be an effective delivery system for kartogenin and served as a promising tissue engineered scaffold for tracheal cartilage regeneration.

Objective: A recent report identified the small molecule kartogenin as a chondrogenic and chondroprotective agent. Since changes in hyaluronan metabolism occur during cartilage degeneration in osteoarthritis, we began studies to determine whether there was a connection between extracellular hyaluronan, CD44–hyaluronan interactions and the effects of kartogenin on articular chondrocytes. Methods: Chondrocytes cultured in monolayers, bioengineered neocartilages, or cartilage explants were treated with kartogenin with or without stimulation by IL-1β. Accumulation of matrix was visualized by a particle exclusion assay or by safranin O staining and release of sulfated glycosaminoglycans was determined. Production of aggrecanases and aggrecan G1-ITEGE neoepitope, fragmentation of CD44 and the SMAD1/5/8 signaling pathway were evaluated by western blotting. Results: Kartogenin treatment enhanced chondrocyte pericellular matrix assembly and retention in the presence of IL-1β. The chondroprotective effects of kartogenin on IL-1β-induced release of sulfated glycosaminoglycans from articular cartilage explants, reduction in safranin O staining of neocartilage discs as well as a reduction in aggrecan G1-ITEGE neoepitope in chondrocyte and explant cartilage cultures were observed. Kartogenin partially blocked the IL-1β-induced increased expression of ADAMTS-5. Additionally, kartogenin-treated articular chondrocytes exhibited a decrease in CD44 proteolytic fragmentation. However, kartogenin treatment did not enhance proteoglycan in control, non-IL-1β-treated cultures. Similarly, kartogenin enhanced the SMAD1 phosphorylation but only following pretreatment with IL-1β. Conclusion: These studies provide novel information on the chondroprotective function of kartogenin in adult articular cartilage. The effects of kartogenin are significant after activation of chondrocytic chondrolysis, which may occur following disruption of homeostasis maintained by hyaluronan–CD44 interactions.

Cytokines are the main cause of intervertebral disc degeneration. Kartogenin (KGN) is found to protect chondrocytes from cytokines. To explore whether KGN can slow down the degeneration on intervertebral discs following exposure to interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF‑α), the expression of type II collagen (Col II) and aggrecan were detected by immunofluorescence, immunohistochemistry and tissue staining. An in vitro model of disc degeneration using human nucleus pulposus cells (hNPCs) and ex vivo culture of mouse intervertebral discs organs under the actions of inflammatory cytokines were used, and the expression of Col II and aggrecan in hNPCs were detected by semi-quantitative western blot analysis, and the mRNA expression of the genes than encode Col II and aggrecan were detected by reverse transcription‑quantitative polymerase chain reaction (RT-qPCR). The results indicated that the expression of Col II and aggrecan was reduced in the degeneration models. However, the protein expressions of Col II and aggrecan were significantly elevated in hNPCs and the mouse intervertebral discs following addition of KGN. RT-qPCR results revealed that the mRNA expression of Col II and aggrecan was increased in hNPCs and mouse intervertebral discs following treatment with KGN. Thus, KGN effectively increased the expression of Col II and aggrecan in hNPCs and slowed the degeneration of intervertebral discs stimulated by IL-1β and TNF-α.

Category: Hindfoot Introduction/Purpose: Entheses have a special fibrocartilage transition zone where tendons and ligaments attach to bone. Enthesis injury is very common, and the reattachment of tendon to bone is a great challenge because healing takes place between a soft tissue (tendon) and a hard tissue (bone). We have now developed a kartogene (KGN)-containing polymer scaffold (KGN-P) that can precisely deliver KGN to damaged enthesis area. The effects of the KGN-containing polymer on the healing of wounded TBJ were investigated in vitro and in vivo. Methods: The proliferation and chondrogenesis of rat Achilles tendon stem cells (TSCs) grown in four conditions were measured: normal medium (Control); normal medium with 100 nM KGN (KGN); lysine diisocyanate (LDI)-glycerol scaffold with normal medium (LDI-P); LDI-glycerol-KGN scaffold with normal medium (KGN-P).A wound (1 mm) was created on each hind leg Achilles enthesis of all 8 rats (3 months old). The wounds were then treated either with 10 ul saline (Wound); or 10 ul of 10 uM KGN (KGN); or LDI polymer scaffold (LDI-P); or KGN-containing polymer scaffold (KGN-P). The rats were sacrificed on day 15 and 30 post-surgery, and their Achilles entheses were collected for gross inspection and histochemical analysis. Results: KGN-containing polymers have sponge-like structures (Fig. 1A-D), and release KGN in a time- and temperature-dependent manner (Fig. 1E). KGN-P scaffold induced chondrogenesis of TSCs (Fig. 2D, 2H) without changing cell proliferation (Fig. 2I), and enhanced fibrocartilage-like tissue formation (Fig. 3E). KGN (Fig. 3C) and LDI-P (Fig. 3D) treated groups exhibited unhealed wound areas as in saline group (Fig. 3B). Finally, KGN-P and KGN treated rat TSCs underwent chondrogenesis by upregulating collagen II, aggrecan, and SOX-9 expression (Fig. 3F). Conclusion: Our results showed that KGN-containing polymer scaffold enhanced wounded enthesis healing by inducing TSC chondrogenesis and promoting the formation of the fibrocartilage in the wound site. The KGN-P may be used for regeneration of wounded entheses in clinical settings. Future research will focus on optimizing KGN concentration and releasing rate in the polymer scaffold during enthesis healing.

Drug Delivery An improved remedy for osteoarthritis could involve localized delivery of an agent to the affected joints to reduce the disease progression, rather than the current use of systemic or local delivery of drugs that only treat the symptoms. Maudens et al. explored the potential for long-term delivery of kartogenin, a pathway activator that enhances articular cartilage protection, by combining two ideas for slow drug release. Using wet milling, they prepared nanocrystals of kartogenin, which dissolve over time, and then embedded them in biodegradable polymer microparticles. The drug-loaded particles were tested in vitro with human synoviocytes and in vivo in a mouse model, in which prolonged delivery and bioactivity of kartogenin were observed. Small 10.1002/smll.201703108 (2018).

Articular cartilage has limited capacity for natural regeneration and repair. In the present study, we evaluated kartogenin (KGN), a bioactive small heterocyclic molecule, for its effect on in vitro proliferation and chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells (hBMSCs) in monolayer culture and in co-culture models in vitro. OA osteochondral cylinders and hBMSCs were collected during total knee replacement. The effect of KGN on hBMSCs during 21 days of culture was monitored by real-time proliferation assay, immunofluorescence staining, histological assay, scanning electron microscopy (SEM) (imaging and multiplex enzyme-linked immunosorbent assay) ELISA assay. The rate of proliferation of hBMSCs was significantly increased by treatment with 10 µM KGN during nine days of culture. Histological and SEM analyses showed the ability of hBMSCs in the presence of KGN to colonize the surface of OA cartilage and to produce glycosaminoglycans and proteoglycans after 21 days of co-culture. KGN treated hBMSCs secreted higher concentrations of TIMPs and the secretion of pro-inflammatory molecules (MMP 13, TNF-α) were significantly suppressed in comparison with control without hBMSCs. Our preliminary results support the concept that 10 µM KGN enhances proliferation and chondrogenic differentiation of hBMSCs and suggest that KGN is a potential promoter for cell-based therapeutic application for cartilage regeneration.

We developed an intra-articular (IA) drug delivery system to treat osteoarthritis (OA) that consisted of kartogenin conjugated chitosan (CHI-KGN). Kartogenin, which promotes the selective differentiation of mesenchymal stem cells (MSCs) into chondrocytes, was conjugated with low-molecular-weight chitosan (LMWCS) and medium-molecular-weight chitosan (MMWCS) by covalent coupling of kartogenin to each chitosan using an ethyl(dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) catalyst. Nanoparticles (NPs, 150 ± 39 nm) or microparticles (MPs, 1.8 ± 0.54 μm) were fabricated from kartogenin conjugated-LMWCS and -MMWCS, respectively, by an ionic gelation using tripolyphosphate (TPP). The in vitro release profiles of kartogenin from the particles showed sustained release for 7 weeks. When the effects of the CHI-KGN NPs or CHI-KGN MPs were evaluated on the in vitro chondrogenic differentiation of human bone marrow MSCs (hBMMSCs), the CHI-KGN NPs and CHI-KGN MPs induced higher expression of chondrogenic markers from cultured hBMMSCs than unconjugated kartogenin. In particular, hBMMSCs treated with CHI-KGN NPs exhibited more distinct chondrogenic properties in the long-term pellet cultures than those treated with CHI-KGN MPs. The in vivo therapeutic effects of CHI-KGN NPs or CHI-KGN MPs were investigated using a surgically-induced OA model in rats. The CHI-KGN MPs showed longer retention time in the knee joint than the CHI-KGN NPs after IA injection in OA rats. The rats treated with CHI-KGN NPs or CHI-KGN MPs by IA injection showed much less degenerative changes than untreated control or rats treated with unconjugated kartogenin. In conclusion, CHI-KGN NPs or CHI-KGN MPs can be useful polymer-drug conjugates as an IA drug delivery system to treat OA.

Category: Basic Sciences/Biologics Introduction/Purpose: The normal tendon-bone junction (TBJ) is a strong structure protected by the fibrocartilage transition zone. This allows a gradual transition of mechanical forces between tendon and bone, thus decreasing stress-concentration effects. Healing of the TBJ interface after an injury is slow and even after healing the junction often lacks the transition zone. A study on human patients showed that even years after ACL reconstruction, patients had no fibrocartilage zone regeneration. This and other studies show that surgical repair alone does not restore the unique protective fibrocartilage transition zone. We have shown that KGN injection into injured rat Achilles tendon-bone junctions enhanced wound healing with restored fibrocartilage transition zone. Here, we examined the effects of KGN treatment, along with platelet-rich plasma (PRP), on tendon-bone tunnel healing in rats. Methods: KGN stock was prepared in DMSO and diluted to 100 μM with PRP. PRP was obtained from the blood of Sprague– Dawley rats and the platelet concentration in PRP was adjusted to 3 times over the baseline platelet concentration in whole blood. Thrombin (1 kU/mL), served as the PRP activator. 27 female rats (234˜268g) were used. A 1.5 mm tunnel was drilled at the distal end of tibia. Achilles tendon was resected and sutured in the tunnel. Rats were randomly divided into 1 of 3 treatment groups: Group A: 50µl KGN + PRP; Group B: 50µl PRP solution; Group C: control. Rats were sacrificed at 4, 8, and 12 weeks for histological analysis. Whole tibia with the tendon insertion were harvested, fixed in 10% formalin and decalcified in 10% EDTA. Tissue were sectioned and stained with Safranin O + Fast green, and immunostained for collagen types 1 and 2. Results: All animals were in good condition after surgery and complications were not present. Safranin O staining was higher in the KGN+PRP group than the other groups indicating more cartilage-like tissues regeneration in this group. Formation of the cartilage-like transitional zone was time dependent; i.e., it increased with increase in time (Fig. 1A-C). In contrast, both PRP and control groups had no cartilage-like tissues (Fig. 1D-I); in fact, some gaps in the control group were found in the tendon-bone interface after 4 and 8 weeks (Fig. 1G-I). Finally, the cartilage-like tissues in the KGN+PRP group also stained positive for both Col-1 and Col-2 indicating that these were fibrocartilage tissues (Fig. 2). Conclusion: Here we demonstrated that KGN promotes the formation of fibrocartilage-like interface between the tendon graft and bone tunnel. This result suggests that the delivery of KGN into the tendon-bone interface could be a promising, cell-free approach to augment the tendon-bone interface healing. PRP in this study, while not effective in promoting fibrocartilage formation of the interface in its own right, it functions as an effective carrier that supplies scaffolds and growth factors necessary for the enhancement of wound healing. Future research is required to determine the optimal KGN dosage regimens and the optimal delivery method (e.g. injection vs implantation).

Category: Basic Sciences/Biologics Introduction/Purpose: Tendon-bone junction (TBJ) injuries are very common and optimal treatment modalities are lacking. TBJ injuries often heal without formation of the fibrocartilage transition zone increasing the risk of rerupture. Current treatments only attach tendons to bones but do not promote healing or regeneration of the fibrocartilage zone. Therefore, we developed a novel approach to heal wounded TBJ using a small molecule called kartogenin (KGN). KGN was previously shown to induce chondrogenesis of tendon stem/progenitor cells (TSCs) in vitro and enhance wound healing in injured rat TBJs in vivo after short- term treatment (2 weeks). It is not known whether these effects can be maintained longterm. Therefore, we studied the long term (3 months) effects of KGN on achilles-tendon-bone junction healing. Methods: KGN stock was prepared in DMSO and diluted to 100 μM with PRP solution. PRP was obtained from the blood of Sprague–Dawley rats and the platelet concentration in PRP was adjusted to 3 times higher than that in whole blood. Thrombin (10 kU/ml) served as the PRP activator. 42 female rats (2.5 – 3 months old) were used. 1 mm diameter defects were created at the Achilles tendon-bone junction area in each hind leg of all rats. Rats were divided into 3 treatment groups: Group 1 (KGN+PRP): KGN (100 μM) +25 μl PRP + 5 μl thrombin; Group 2 (PRP): 25 μl PRP + 5 μl thrombin; and Group 3 (Control): 30 μl saline. All the rats were sacrificed at 3 months, and hind legs harvested for analyses. N= 6 hind legs were used for histological and qPCR analysis. N = 16 hind legs were used for mechanical testing. Results: Single treatment of KGN+PRP demonstrates improved TBJ healing (Fig. 1A) compared to PRP only and control groups (Fig. 1B, C). Fibrocartilage-like tissue regeneration occurred only in the KGN+PRP treated group (Fig. 2A). These results were further confirmed by qPCR, which showed significantly higher expression of cartilage-related genes (Data not shown). Furthermore, the cartilage-like transition zone in the KGN+PRP group also stained positive for Col-I, Col-II, Scx and Sox-9 indicating the regeneration of fibrocartilage tissues in the KGN+PRP treated TBJ (Fig. 3A, B). Mechanical testing showed that the KGN+PRP (47±12 N) and PRP (50±13 N) groups had significantly higher ultimate strength than the controls (39±16 N). There was no significant difference in ultimate strength between the KGN+PRP group and PRP group. Conclusion: In this study, we have shown that KGN injection enhanced healing of the wounded TBJ by promoting the formation of a fibrocartilage transition zone. Thus, KGN can be used as a mode of cell free therapy to promote regeneration of the fibrocartilage transition zone in injured TBJs. PRP alone enhanced healing of the TBJ but did not induce fibrocartilage regeneration. PRP + KGN are advantageous due to the numerous growth factors located within PRP and PRPs ability to serve as a KGN carrier. Future research is needed to optimize KGN and PRP dosage regimens and the optimal delivery methods.

Limb development requires the coordinated growth of several tissues and structures including long bones, joints and tendons, but the underlying mechanisms are not wholly clear. Recently, we identified a small drug-like molecule – we named Kartogenin (KGN) – that greatly stimulates chondrogenesis in marrow-derived mesenchymal stem cells (MSCs) and enhances cartilage repair in mouse osteoarthritis (OA) models. To determine whether limb developmental processes are regulated by KGN, we tested its activity on committed preskeletal mesenchymal cells from mouse embryo limb buds and whole limb explants. KGN did stimulate cartilage nodule formation and more strikingly, boosted digit cartilaginous anlaga elongation, synovial joint formation and interzone compaction, tendon maturation as monitored by ScxGFP, and interdigit invagination. To identify mechanisms, we carried out gene expression analyses and found that several genes, including those encoding key signaling proteins, were up-regulated by KGN. Amongst highly up-regulated genes were those encoding hedgehog and TGFβ superfamily members, particularly TFGβ1. The former response was verified by increases in Gli1-LacZ activity and Gli1 mRNA expression. Exogenous TGFβ1 stimulated cartilage nodule formation to levels similar to KGN, and KGN and TGFβ1 both greatly enhanced expression of lubricin/Prg4 in articular superficial zone cells. KGN also strongly increased the cellular levels of phospho-Smads that mediate canonical TGFβ and BMP signaling. Thus, limb development is potently and harmoniously stimulated by KGN. The growth effects of KGN appear to result from its ability to boost several key signaling pathways and in particular TGFβ signaling, working in addition to and/or in concert with the filamin A/CBFβ/RUNX1 pathway we identified previously to orchestrate overall limb development. KGN may thus represent a very powerful tool not only for OA therapy, but also limb regeneration and tissue repair strategies.

Introduction Microfracture does not properly repair full-thickness cartilage defects. The purpose of this study was to evaluate the effect of intraarticular injection of the small-molecule compound kartogenin (KGN) on the restoration of a full-thickness cartilage defect treated with microfracture in a rabbit model. Methods Full-thickness cartilage defects (3.5 mm in diameter and 3 mm in depth) were created in the patellar groove of the right femurs of 24 female New Zealand White rabbits. The rabbits were divided into two groups (12 in each group) based on postsurgery treatment differences, as follows: microfracture plus weekly intraarticular injection of KGN (group 1) and microfracture plus dimethyl sulfoxide (group 2). Six rabbits from each group were illed at 4 and 12 weeks after surgery, and their knees were harvested. The outcome was assessed both macroscopically, by using the International Cartilage Repair Society (ICRS) macroscopic evaluation system, and histologically, by using the modified O’Driscoll histologic scoring system. Immunohistochemistry for type II and I collagen was also conducted. Results At 4 weeks, group 1 showed better defect filling and a greater number of chondrocyte-like cells compared with group 2. At 12 weeks, group 1 showed statistically significantly higher ICRS scores and modified O’Driscoll scores compared with group 2. More hyaline cartilage-like tissue was found in the defects of group 1 at 12 weeks. Conclusions Intraarticular injection of KGN enhances the quality of full-thickness cartilage defects repair after microfracture, with better defect filling and increased hyaline-like cartilage formation.

Biological therapy is a thriving area of research and development, and is well established for chronic forms of rheumatoid arthritis (RA) and ankylosing spondylitis (AS). However, there is no clinically validated biological therapy for osteoarthritis (OA). Chronic forms of OA are increasingly viewed as an inflammatory disease. OA was largely regarded as a “wear and tear disease”. However, the disease is now believed to involve “low grade” inflammation and the growth of blood vessels and nerves from the subchondral bone into articular cartilage. This realization has focused research effort on the development and evaluation of biological therapy that targets proinflammatory mediators, angiogenic factors and cytokines in articular cartilage, subchondral bone and synovium in chronic forms of OA. This review article provides an overview of emerging biological therapy for OA, and discusses recent molecular targets implicated in angiogenesis and neurogenesis and progress with antibody-based therapy, calcitonin, and kartogenin, the small molecule stimulator of chondrogenesis.