Spatially unique Shh-Fgf8 distribution in regenerating limb guarantees consistent limb morphogenesis in different limb sizes and contributes to axolotl specific digit patterning.

Limb regeneration in Ambystoma mexicanum occurs in various sizes of fields and can recreate consistent limb morphology. It was not known what mechanism supports such stable limb morphogenesis regardless of size. Limb regeneration in urodele amphibians has been basically considered to recapitulate the limb developmental processes. Many molecules in the limb developmental processes are conserved with other tetrapods. SHH and FGF8 play important roles in the morphogenesis of limbs among them. Focusing on these two factors, we investigated the detailed expression pattern of Shh and Fgf8 in the various sizes of blastema in axolotl limb regeneration. Fgf8 is expressed in the anterior side of a blastema and Shh is expressed in the posterior side. These are maintained in a mutually dependent manner. We also clarified that the size of Shh and Fgf8 expression domains were scaled as the size of the blastemas increased. However, it was found that the secretion and working range of SHH were kept constant. We also found that the consistent SHH secretion range contributed to promoting cell proliferation and the first digital cartilage differentiation near the Shh expression domain. This would be a reasonable system to guarantees constant limb morphogenesis regardless of the blastema size. We also showed that the Shh-Fgf8 expression domain was shifted posteriorly as the digital differentiation progressed. Consistently, slowing the timing of blocking Shh signaling resulted in morphological defects that could be observed in only posterior digits. The revealed posteriorly shifting Shh-Fgf8 domain might explain urodele specific digit formation, in which digits are added posteriorly.

Introducing dorsoventral patterning in adult regenerating lizard tails with gene-edited embryonic neural stem cells

Lizards regenerate amputated tails but fail to recapitulate the dorsoventral patterning achieved during embryonic development. Regenerated lizard tails form ependymal tubes (ETs) that, like embryonic tail neural tubes (NTs), induce cartilage differentiation in surrounding cells via sonic hedgehog (Shh) signaling. However, adult ETs lack characteristically roof plate-associated structures and express Shh throughout their circumferences, resulting in the formation of unpatterned cartilage tubes. Both NTs and ETs contain neural stem cells (NSCs), but only embryonic NSC populations differentiate into roof plate identities when protected from endogenous Hedgehog signaling. NSCs were isolated from parthenogenetic lizard embryos, rendered unresponsive to Hedgehog signaling via CRISPR/Cas9 gene knockout of smoothened (Smo), and implanted back into clonally-identical adults to regulate tail regeneration. Here we report that Smo knockout embryonic NSCs oppose cartilage formation when engrafted to adult ETs, representing an important milestone in the creation of regenerated lizard tails with dorsoventrally patterned skeletal tissues.

Injectable stress relaxation gelatin-based hydrogels with positive surface charge for adsorption of aggrecan and facile cartilage tissue regeneration

Background Cartilage injury and pathological degeneration are reported in millions of patients globally. Cartilages such as articular hyaline cartilage are characterized by poor self-regeneration ability due to lack of vascular tissue. Current treatment methods adopt foreign cartilage analogue implants or microfracture surgery to accelerate tissue repair and regeneration. These methods are invasive and are associated with the formation of fibrocartilage, which warrants further exploration of new cartilage repair materials. The present study aims to develop an injectable modified gelatin hydrogel. Method The hydrogel effectively adsorbed proteoglycans secreted by chondrocytes adjacent to the cartilage tissue in situ, and rapidly formed suitable chondrocyte survival microenvironment modified by ε-poly-L-lysine (EPL). Besides, dynamic covalent bonds were introduced between glucose and phenylboronic acids (PBA). These bonds formed reversible covalent interactions between the cis−diol groups on polyols and the ionic boronate state of PBA. PBA-modified hydrogel induced significant stress relaxation, which improved chondrocyte viability and cartilage differentiation of stem cells. Further, we explored the ability of these hydrogels to promote chondrocyte viability and cartilage differentiation of stem cells through chemical and mechanical modifications. Results In vivo and in vitro results demonstrated that the hydrogels exhibited efficient biocompatibility. EPL and PBA modified GelMA hydrogel (Gel-EPL/B) showed stronger activity on chondrocytes compared to the GelMA control group. The Gel-EPL/B group induced the secretion of more extracellular matrix and improved the chondrogenic differentiation potential of stem cells. Finally, thus hydrogel promoted the tissue repair of cartilage defects. Conclusion Modified hydrogel is effective in cartilage tissue repair.

A Gelatin-Hyaluronic Acid Double Cross-Linked Hydrogel for Regulating the Growth and Dual Dimensional Cartilage Differentiation of Bone Marrow Mesenchymal Stem Cells

Owing to its unique physiochemical properties similar to the extracellular matrix (ECM), three-dimensional (3D) crosslinked hydrogels are widely studied materials for tissue engineering. In this study, to mimic the ECM microenvironment, a two-step covalent cross-linking with hyaluronic acid and gelatin was performed to form an interpenetrating polymer network structure. Gelatin as the first network greatly improved the mechanical strength of the hydrogels, while a hyaluronic acid network as the second network improved the tenacity and biological activity. Compared with a single network hydrogel, the interpenetrating hydrogel system can further regulate the mechanical properties of the hydrogel by adjusting the ratio of the two components, thereby changing the proliferation, activity, and direction of cartilage differentiation of bone marrow mesenchymal stem cells (BMSCs). Not only that, with two culture methods for BMSCs on the surface and 3D wrapped in the double cross-linked hydrogels, they exhibited their potential to induce BMSCs to cartilage differentiation under the condition of 3D encapsulation of BMSCs and contact with BMSCs on its surface. As a scaffold material for cartilage tissue engineering, this double cross-linked hydrogel demonstrated its high feasibility and applicability in delivering BMSCs in vivo and repairing defects.

PRDM proteins control Wnt/β-catenin activity to regulate craniofacial chondrocyte differentiation

Cranial neural crest (NCC)-derived chondrocyte precursors undergo a dynamic differentiation and maturation process to establish a scaffold for subsequent bone formation, alterations in which contribute to congenital birth defects. Here, we demonstrate that transcription factor and histone methyltransferase proteins Prdm3 and Prdm16 control the differentiation switch of cranial NCCs to craniofacial cartilage. Loss of either results in hypoplastic and unorganized chondrocytes due to impaired cellular orientation and polarity. We show that PRDMs regulate cartilage differentiation by controlling the timing of Wnt/β-catenin activity in strikingly different ways: Prdm3 represses while Prdm16 activates global gene expression, though both by regulating Wnt enhanceosome activity and chromatin accessibility. Finally, we show that manipulating Wnt/β-catenin signaling pharmacologically or generating prdm3-/-;prdm16-/- double mutants rescues craniofacial cartilage defects. Our findings reveal upstream regulatory roles for Prdm3 and Prdm16 in cranial NCCs to control Wnt/β-catenin transcriptional activity during chondrocyte differentiation to ensure proper development of the craniofacial skeleton.

HGH and Cartilage

Recombinant human growth hormone (rHGH) which plays an important role for remodeling of bone has an effect on cartilage differentiation and regeneration, as well. Herein, we reviewed human growth hormone and its roles on cartilage. We discussed different roles on growth, regeneration, inflammation, cell maturation, and mitotic activity. These findings provide proof of principle that therapeutics based on rHGH can improve treatment for numerous disorders.

Unbiased transcriptomic analysis of chondrocyte differentiation in a high-density cell culture model

ABSTRACTIn the developing vertebrate skeleton, cartilage is an important precursor to the formation of bones. Cartilage is produced by chondrocytes, which derive from embryonic mesoderm and undergo a stereotypical program of differentiation and maturation. Here we modeled this process in vitro, using primary fetal mouse rib chondrocytes in a high-density cell culture model of cartilage differentiation, and performed genome-wide gene expression profiling over the course of culture. The overarching goal of this study was to characterize the molecular pathways involved in cartilage differentiation and maturation. Our results also enable a comprehensive appraisal of distinctions between common in vitro models for cartilage differentiation, and of differences in their molecular resemblance to cartilage formation in vivo.

BMP signaling is necessary and sufficient for osteoarthritis and a target for disease modifying therapy

Osteoarthritis (OA) is among the leading causes of disability across the world. Presently no effective therapy of OA is available as neither the molecular mechanism of disease pathology nor the development and maintenance of articular cartilage is well understood. During OA, articular cartilage undergoes cellular and molecular changes reminiscent of transient cartilage, which is the embryonic precursor of endochondral bone. During endochondral ossification, a precise spatio-temporally regulated WNT-BMP signaling interplay dictates differentiation of a common progenitor pool to either articular or transient cartilage fate in adjacent domains. In the embryonic context, Wnt signaling promotes articular cartilage fate while transient cartilage differentiation is critically BMP signaling dependent. Moreover, any ectopic activation of BMP signaling leads to ectopic transient cartilage differentiation at the expense of articular cartilage. In this study, we show that BMP signaling is sufficient and necessary for the pathogenesis of OA by ectopically activating BMP signaling and depleting BMP ligands in adult mice articular cartilage, respectively. Analysis of human osteoarthritic specimens demonstrated association between OA and ectopic BMP signaling in the articular cartilage. Local inhibition of BMP signaling using a potent pharmacological inhibitor LDN-193189, when administered prophylactically, resulted in delayed onset and reduced severity of OA in mice. Additionally, the same treatment afforded protection against cartilage degeneration post onset of OA in a surgical model of OA in mice. Therefore, inhibiting BMP signaling and consequent block of transient cartilage differentiation within the cells of the joint cartilage can be a possible avenue for developing a disease modifying therapy for OA.