Skeletal Defects Research Articles

Zebrafish as a model to study PERK function in developmental diseases: implications for Wolcott-Rallison syndrome.

Developmental diseases are challenging to investigate due to their clinical heterogeneity and relatively low prevalence. The Wolcott-Rallison Syndrome (WRS) is a rare developmental disease characterized by skeletal dysplasia and permanent neonatal diabetes due to loss-of-function mutations in the endoplasmic reticulum stress kinase PERK (EIF2AK3). The lack of efficient and less invasive therapies for WRS highlights the need for new animal models that replicate the complex pathological phenotypes, while preserving scalability for drug screening. Zebrafish exhibits high fecundity and rapid development that facilitate efficient and scalable in vivo drug testing. Here, we aimed to assess the potential of zebrafish to study PERK function and its pharmacological modulation, and as model organism of developmental diseases such as the WRS. Using bioinformatic analyses, we showed high similarity between human and zebrafish PERK. We used the pharmacological PERK inhibitor GSK2606414, which was bioactive in zebrafish, to modulate PERK function. Using transgenic zebrafish expressing fluorescent pancreatic markers and a fluorescent glucose probe, we observed that PERK inhibition decreased β cell mass and disrupted glucose homeostasis. By combining behavioural and functional assays, we show that PERK-inhibited zebrafish present marked skeletal defects and defective growth, as well as neuromuscular and cardiac deficiencies, which are clinically relevant in WRS patients, while sparing parameters like otolith area and eye/body ratio which are not associated with WRS. These results show that zebrafish holds potential to study PERK function and its pharmacological modulation in developmental disorders like WRS, assisting research on their pathophysiology and experimental treatments.

Extracellular matrix-mimicking cryogels composed of methacrylated fucoidan enhance vascularized skeletal muscle regeneration following volumetric muscle loss

Volumetric muscle loss (VML) significantly impairs the inherent regenerative ability of skeletal muscle and results in chronic functional impairment. Polysaccharides in the muscle extracellular matrix are crucial for regulating cell proliferation and differentiation. Recent studies indicate that fucoidan has beneficial effects on musculoskeletal conditions. However, the impact of fucoidan on skeletal muscle regeneration remains poorly understood. In this study, methacrylated fucoidan (FuMA) was synthesized through chemical grafting of the methacryloyl group onto fucoidan. In vitro experiments demonstrated that treatment with FuMA significantly up-regulated the expression of myogenic markers and promoted the formation of myotubes in C2C12 myoblast cells. Importantly, FuMA treatment led to a significant enhancement in mitochondrial energy metabolism of myoblasts via activation of the NRF2 antioxidant signaling pathway. To further investigate the regenerative properties in repairing skeletal muscle defects, we fabricated a dual crosslinked cryogel consisting of FuMA and methacrylated gelatin (GelMA) with a porous and interconnected structure. In a rat tibialis anterior muscle VML model, implantation of the FuMA/GelMA cryogel effectively promoted the regeneration of muscle fibers, reduced collagen deposition, and facilitated the formation of new blood vessels. Hence, polysaccharide-based cryogels represent a promising implantable biomimetic scaffold for facilitating skeletal muscle regeneration following severe injuries.

Signaling Pathways Concerning Mitochondrial Dysfunction: Implications in Neurodegeneration and Possible Molecular Targets.

Mitochondrion is an important organelle present in our cells responsible for meeting energy requirements. All higher organisms rely on efficient mitochondrial bioenergetic machinery to sustain life. No other respiratory process can produce as much power as generated by mitochondria in the form of ATPs. This review is written in order to get an insight into the magnificent working of mitochondrion and its implications in cellular homeostasis, bioenergetics, redox, calcium signaling, and cell death. However, if this machinery gets faulty, it may lead to several disease states. Mitochondrial dysfunctioning is of growing concern today as it is seen in the pathogenesis of several diseases which includes neurodegenerative disorders, cardiovascular disorders, diabetes mellitus, skeletal muscle defects, liver diseases, and so on. To cover all these aspects is beyond the scope of this article; hence, our study is restricted to neurodegenerative disorders only. Moreover, faulty functioning of this organelle can be one of the causes of early ageing in individuals. This review emphasizes mutations in the mitochondrial DNA, defects in oxidative phosphorylation, generation of ROS, and apoptosis. Researchers have looked into new approaches that might be able to control mitochondrial failure and show a lot of promise as treatments.

Highly porous polycaprolactone microspheres for skeletal repair promote a mature bone cell phenotype in vitro.

Improving our ability to treat skeletal defects is a critical medical challenge that necessitates the development of new biomaterials. One promising approach involves the use of degradable polymer microparticles with an interconnected internal porosity. Here, we employed a double emulsion to generate such round microparticles (also known as microspheres) from a polycaprolactone-based polymerised high internal phase emulsion (polyHIPE). These microspheres effectively supported the growth of mesenchymal progenitors over a 30-day period, and when maintained in osteogenic media, cells deposited a bone-like extracellular matrix, as determined by histological staining for calcium and collagen. Interestingly, cells with an osteocyte-like morphology were observed within the core of the microspheres indicating the role of a physical environment comparable to native bone for this phenotype to occur. At later timepoints, these cultures had significantly increased mRNA expression of the osteocyte-specific markers dentin matrix phosphoprotein-1 (Dmp-1) and sclerostin, with sclerostin also observed at the protein level. Cells pre-cultured on porous microspheres exhibited enhanced survival rates compared to those pre-cultured on non-porous counterparts when injected. Cells precultured on both porous and non-porous microspheres promoted angiogenesis in a chorioallantoic membrane (CAM) assay. In summary, the polycaprolactone polyHIPE microspheres developed in this study exhibit significant promise as an alternative to traditional synthetic bone graft substitutes, offering a conducive environment for cell growth and differentiation, with the potential for better clinical outcomes in bone repair and regeneration.

Macrophage-derived amphiregulin promoted the osteogenic differentiation of chondrocytes through EGFR/Yap axis and TGF-β activation

Endochondral ossification represents a crucial biological process in skeletal development and bone defect repair. Macrophages, recognized as key players in the immune system, are now acknowledged for their substantial role in promoting endochondral ossification within cartilage. Concurrently, the epidermal growth factor receptor (EGFR) ligand amphiregulin (Areg) has been documented for its contributory role in restoring bone tissue homeostasis post-injury. However, the mechanism by which macrophage-secreted Areg facilitates bone repair remains elusive. In this study, the induction of macrophage depletion through in vivo administration of clodronate liposomes was employed in a standard open tibial fracture mouse model to assess bone healing using micro-computed tomography (micro-CT) analysis, histomorphology, and ELISA serum evaluations. The investigation revealed sustained expression of Areg during the fracture healing period in wild-type mice. Macrophage depletion significantly reduced the number of macrophages on the local bone surface and vital organs. This reduction led to diminished Areg secretion, decreased collagen production, and delayed fracture healing. However, histological and micro-CT assessments at 7 and 21 days post-local Areg treatment exhibited a marked improvement of bone healing compared to the vehicle control. In vitro studies demonstrated an increase of Areg secretion by the Raw264.7 cells upon ATP stimulation. Indirect co-culture of Raw264.7 and ATDC5 cells indicated that Areg overexpression enhanced the osteogenic potential of chondrocytes, and vice versa. This osteogenic promotion was attributed to Areg's activation of the membrane receptor EGFR in the ATDC5 cell line, the enhanced phosphorylation of transcription factor Yap, and the facilitation of the expression of bioactive TGF-β by chondrocytes. Collectively, this research elucidates the direct mechanistic effects of macrophage-secreted Areg in promoting bone homeostasis following bone injury.

Mechanical Stimulation and Aligned Poly(ε-caprolactone)-Gelatin Electrospun Scaffolds Promote Skeletal Muscle Regeneration.

The current treatments to restore skeletal muscle defects present several injuries. The creation of scaffolds and implant that allow the regeneration of this tissue is a solution that is reaching the researchers' interest. To achieve this, electrospinning is a useful technique to manufacture scaffolds with nanofibers with different orientation. In this work, polycaprolactone and gelatin solutions were tested to fabricate electrospun scaffolds with two degrees of alignment between their fibers: random and aligned. These scaffolds can be seeded with myoblast C2C12 and then stimulated with a mechanical bioreactor that mimics the physiological conditions of the tissue. Cell viability as well as cytoskeletal morphology and functionality was measured. Myotubes in aligned scaffolds (9.84 ± 1.15 μm) were thinner than in random scaffolds (11.55 ± 3.39 μm; P = 0.001). Mechanical stimulation increased the width of myotubes (12.92 ± 3.29 μm; P < 0.001), nuclear fusion (95.73 ± 1.05%; P = 0.004), and actin density (80.13 ± 13.52%; P = 0.017) in aligned scaffolds regarding the control. Moreover, both scaffolds showed high myotube contractility, which was increased in mechanically stimulated aligned scaffolds. These scaffolds were also electrostimulated at different frequencies and they showed promising results. In general, mechanically stimulated aligned scaffolds allow the regeneration of skeletal muscle, increasing viability, fiber thickness, alignment, nuclear fusion, nuclear differentiation, and functionality.

A human progeria-associated BAF-1 mutation modulates gene expression and accelerates aging in C. elegans.

Alterations in the nuclear envelope are linked to a variety of rare diseases termed laminopathies. A single amino acid substitution at position 12 (A12T) of the human nuclear envelope protein BAF (Barrier to Autointegration Factor) causes Néstor-Guillermo Progeria Syndrome (NGPS). This premature ageing condition leads to growth retardation and severe skeletal defects, but the underlying mechanisms are unknown. Here, we have generated a novel in vivo model for NGPS by modifying the baf-1 locus in C. elegans to mimic the human NGPS mutation. These baf-1(G12T) mutant worms displayed multiple phenotypes related to fertility, lifespan, and stress resistance. Importantly, nuclear morphology deteriorated faster during aging in baf-1(G12T) compared to wild-type animals, recapitulating an important hallmark of cells from progeria patients. Although localization of BAF-1(G12T) was similar to wild-type BAF-1, lamin accumulation at the nuclear envelope was reduced in mutant worms. Tissue-specific chromatin binding andtranscriptome analyses showed reduced BAF-1 association in most genes deregulated by the baf-1(G12T) mutation, suggesting that altered BAF chromatin association induces NGPS phenotypes via altered gene expression.

Loss of Hdac4 in Osteoprogenitors Impairs Postnatal Trabecular and Cortical Bone Formation, Resulting in a Dwarfism and Osteopenia Phenotype in Mice

HDAC4 is a class II histone deacetylation protein with a well-characterized role in chondrocyte differentiation and skeletal development, and dysregulated expression or haploinsufficiency of Hdac4 leads to skeletal formation and malformation disorders. The early lethality of hdac4 ablation mice hindered further investigation of its role in postnatal bone growth and development. Therefore, this study aims to investigate the significant role of Hdac4 in postnatal endochondral bone development using two mouse models with conditional deletion of Hdac4 in Sp7-expressing osteoprogenitors or chondrocytes and monitored postnatal bone development. The phenotype of Acan-CreERT2; Hdac4fl/fl mice largely resembled that of conventional Hdac4-/- mice. But phenotypic characterizations of mice with Hdac4 inactivation in Sp7-expressing osteoprogenitors (Sp7-Cre; Hdac4fl/fl) showed dwarfism with body and limb shortening and remarkable skeletal defects. Micro-computed tomography analysis of tibias further demonstrated that loss of Hdac4 expression impaired bone formation and microarchitecture, mainly characterized by dysplasia of trabecular and cortical bone in young mice. Our in vivo and in vitro data support a crucial role for Hdac4 in regulating osteoblast proliferation and differentiation, bone matrix protein production, angiogenesis, and ultimately trabecular and cortical bone formation. Moreover, RNA-seq analysis implicated Hdac4 in the regulation of key genes and pathways necessary to affect the accumulation of extracellular matrix, biological processes related to signal transduction, and skeletal growth. Collectively, our data show that postnatal expression of Hdac4 in Sp7-expressing osteoprogenitors provides essential regulatory oversight of endochondral bone formation, bone morphology, and homeostasis.

Expanding the Spectrum of Endocrine Abnormalities Associated with SOX11-related Disorders.

SOX11 variants cause Coffin-Siris Syndrome (CSS), characterized by developmental delay, hypogonadotropic hypogonadism (HH), skeletal and facial defects. To examine the contribution of SOX11 variants to the pathogenesis of Idiopathic Hypogonadotropic Hypogonadism (IHH), a disorder caused by hypothalamic GnRH deficiency. The Reproductive Endocrine Unit and the Pediatric Endocrinology Division, Massachusetts General Hospital. A cohort of 1810 unrelated IHH probands. Exome sequencing data from the entire cohort were examined for SOX11 rare single nucleotide variants (SNVs) [minor allele frequency in the gnomAD database <0.1%]. Rare SOX11 variant association testing was performed between the IHH and gnomAD population. Phenotyping of individuals harboring pathogenic/likely pathogenic SNVs (determined by the ACMG criteria) was performed. Four pathogenic SOX11 SNVs were identified in 5 IHH probands. The IHH cohort was enriched for SOX11 protein truncating SNVs (frameshift/nonsense) across the entire protein (2 SNVs in 3 IHH cases [p.S303X (de-novo); p.S345Afs*13]; p 0.0004981) and for SOX11 missense SNVs within the SOX11-high-mobility group (HMG) domain (2 SNVs in 2 IHH cases p.G84D[de-novo]; p.P114S; p=0.00313922). The phenotypic spectrum of SOX11 variant carriers revealed additional endocrine defects including anosmic and normosmic forms of IHH, growth-hormone deficiency, pituitary and hypothalamic structural defects, and hypothyroidism. A pathogenic SOX11 SNV was also identified in a patient with functional HH (FHH, p.R100Q). CSS-associated features were present in 4/5 probands. Deleterious SOX11 variants cause IHH and other pituitary hormone deficiencies, suggesting that the human SOX11-associated disorder may stem from both hypothalamic and pituitary level defects.

Outcomes of Free Vascularized Fibular Grafts in Treating Massive Forearm Skeletal Defects.

Reconstructing long bone defects in the upper limbs, particularly the radius and ulna, poses significant challenges. These defects, resulting from trauma, tumors, infections, or congenital anomalies, require precise surgical intervention for functional restoration. Traditional non-vascularized autogenous bone grafts have limitations, such as resorption and limited biological activity. To address these challenges, free vascularized fibular grafts (FVFGs) have been developed, offering enhanced recovery by supplying nutrients and structural support, particularly in large defects or compromised vascularity. This retrospective study reviewed patients with significant forearm skeletal defects treated with FVFGs at our institution from January 2008 to January 2019. Included were patients with radius or ulna defects exceeding 8 cm due to trauma, tumor excision, or non-union fractures. Data on demographics, clinical details, surgical techniques, and outcomes-including graft union time, complications, range of motion, and the disabilities of the arm, shoulder and hand (DASH) scores-were analyzed. Eight patients, with a mean age of 27.6 years and an average defect length of 9.8 cm, were included. All patients achieved graft union within an average of 4 months, with no tumor recurrence or significant complications. Functional outcomes showed mean forearm pronation of 56.9 degrees, supination of 52.5 degrees, and a mean DASH score of 17.7. FVFG is a safe and effective technique for managing complex forearm bone defects, providing high union rates and good functional outcomes. It should be considered a primary option for large forearm skeletal defects.

Metabolic rewiring during bone development underlies tRNA m7G-associated primordial dwarfism.

Translation of mRNA to protein is tightly regulated by transfer RNAs (tRNAs), which are subject to various chemical modifications that maintain structure, stability, and function. Deficiency of tRNA N7-methylguanosine (m7G) modification in patients causes a type of primordial dwarfism, but the underlying mechanism remains unknown. Here we report that the loss of m7G rewires cellular metabolism, leading to the pathogenesis of primordial dwarfism. Conditional deletion of the catalytic enzyme Mettl1 or missense mutation of the scaffold protein Wdr4 severely impaired endochondral bone formation and bone mass accrual. Mechanistically, Mettl1 knockout decreased abundance of m7G-modified tRNAs and inhibited translation of mRNAs relating to cytoskeleton and Rho GTPase signaling. Meanwhile, Mettl1 knockout enhanced cellular energy metabolism despite incompetent proliferation and osteogenic commitment. Further exploration revealed that impairment of Rho GTPase signaling upregulated the level of branched-chain amino acid transaminase 1 (BCAT1) that rewired cell metabolism and restricted intracellular α-ketoglutarate (αKG). Supplementation of αKG ameliorated the skeletal defect of Mettl1-deficient mice. In addition to the selective translation of metabolism-related mRNAs, we further revealed that Mettl1 knockout globally regulated translation via integrated stress response (ISR) and mammalian target of rapamycin complex 1 (mTORC1) signaling. Restoring translation by targeting either ISR or mTORC1 aggravated bone defects of Mettl1-deficient mice. Overall, our study unveils a critical role of m7G tRNA modification in bone development by regulation of cellular metabolism and indicates suspension of translation initiation as a quality control mechanism in response to tRNA dysregulation.

Impaired Skeletal Development by Disruption of Presenilin-1 in Pigs and Generation of Novel Pig Models for Alzheimer's Disease.

Presenilin 1 (PSEN1) is one of the genes linked to the prevalence of early onset Alzheimer's disease. In mice, inactivation of Psen1 leads to developmental defects, including vertebral malformation and neural development. However, little is known about the role of PSEN1 during the development in other species. To investigate the role of PSEN1 in vertebral development and the pathogenic mechanism of neurodegeneration using a pig model. CRISPR/Cas9 system was used to generate pigs with different mutations flanking exon 9 of PSEN1, including those with a deleted exon 9 (Δexon9). Vertebral malformations in PSEN1 mutant pigs were examined by X-ray, micro-CT and micro-MRI. Neuronal cells from the brains of PSEN1 mutant pigs were analyzed by immunoflourescence, followed by image analysis including morphometric evaluation via image J and 3D reconstruction. Pigs with a PSEN1 null mutation (Δexon9-12) died shortly after birth and had significant axial skeletal defects, whereas pigs carrying at least one Δexon9 allele developed normally and remained healthy. Effects of the null mutation on abnormal skeletal development were also observed in fetuses at day 40 of gestation. Abnormal distribution of astrocytes and microglia in the brain was detected in two PSEN1 mutant pigs examined compared to age-matched control pigs. The founder pigs were bred to establish and age PSEN1ΔE9/+ pigs to study their relevance to clinical Alzheimer's diseases. PSEN1 has a critical role for normal vertebral development and PSEN1 mutant pigs serves as novel resources to study Alzheimer's disease.

SRSF2 is a key player in orchestrating the directional migration and differentiation of MyoD progenitors during skeletal muscle development

SRSF2 plays a dual role, functioning both as a transcriptional regulator and a key player in alternative splicing. The absence of Srsf2 in MyoD + progenitors resulted in perinatal mortality in mice, accompanied by severe skeletal muscle defects. SRSF2 deficiency disrupts the directional migration of MyoD progenitors, causing them to disperse into both muscle and non-muscle regions. Single-cell RNA-sequencing analysis revealed significant alterations in Srsf2-deficient myoblasts, including a reduction in extracellular matrix components, diminished expression of genes involved in ameboid-type cell migration and cytoskeleton organization, mitosis irregularities, and premature differentiation. Notably, one of the targets regulated by Srsf2 is the serine/threonine kinase Aurka. Knockdown of Aurka led to reduced cell proliferation, disrupted cytoskeleton, and impaired differentiation, reflecting the effects seen with Srsf2 knockdown. Crucially, the introduction of exogenous Aurka in Srsf2-knockdown cells markedly alleviated the differentiation defects caused by Srsf2 knockdown. Furthermore, our research unveiled the role of Srsf2 in controlling alternative splicing within genes associated with human skeletal muscle diseases, such as BIN1, DMPK, FHL1, and LDB3. Specifically, the precise knockdown of the Bin1 exon17-containing variant, which is excluded following Srsf2 depletion, profoundly disrupted C2C12 cell differentiation. In summary, our study offers valuable insights into the role of SRSF2 in governing MyoD progenitors to specific muscle regions, thereby controlling their differentiation through the regulation of targeted genes and alternative splicing during skeletal muscle development.

SRSF2 is a key player in orchestrating the directional migration and differentiation of MyoD progenitors during skeletal muscle development.

SRSF2 plays a dual role, functioning both as a transcriptional regulator and a key player in alternative splicing. The absence of Srsf2 in MyoD + progenitors resulted in perinatal mortality in mice, accompanied by severe skeletal muscle defects. SRSF2 deficiency disrupts the directional migration of MyoD progenitors, causing them to disperse into both muscle and non-muscle regions. Single-cell RNA-sequencing analysis revealed significant alterations in Srsf2-deficient myoblasts, including a reduction in extracellular matrix components, diminished expression of genes involved in ameboid-type cell migration and cytoskeleton organization, mitosis irregularities, and premature differentiation. Notably, one of the targets regulated by Srsf2 is the serine/threonine kinase Aurka. Knockdown of Aurka led to reduced cell proliferation, disrupted cytoskeleton, and impaired differentiation, reflecting the effects seen with Srsf2 knockdown. Crucially, the introduction of exogenous Aurka in Srsf2-knockdown cells markedly alleviated the differentiation defects caused by Srsf2 knockdown. Furthermore, our research unveiled the role of Srsf2 in controlling alternative splicing within genes associated with human skeletal muscle diseases, such as BIN1, DMPK, FHL1, and LDB3. Specifically, the precise knockdown of the Bin1 exon17-containing variant, which is excluded following Srsf2 depletion, profoundly disrupted C2C12 cell differentiation. In summary, our study offers valuable insights into the role of SRSF2 in governing MyoD progenitors to specific muscle regions, thereby controlling their differentiation through the regulation of targeted genes and alternative splicing during skeletal muscle development.

Study of Akabane disease in an Iranian dairy herd: a re-emerging disease.

Akabane virus is a teratogenic pathogen transmitted by Culicoides spp. to ruminants. The virus induces anomalies in the central nervous system in the developing fetus, resulting in arthrogryposis-hydranencephaly (A-H) syndrome. During three outbreaks of the disease (2002, 2013, and 2020), 77 calves were born in Varamin, Iran, with A-H syndrome. The presenting neurologic signs were categorized into three main groups, as common, less common, and uncommon signs. The common signs were unawareness of the surroundings, blindness, deep depression, partial failure of suckling, and unintelligent behavior. The less commonly noted signs were hyperexcitability, regurgitation, head pressing, compulsive walking, and kicking, while the uncommon signs comprised protrusion of the tongue, making sounds resembling barking, carnivore-like milk drinking, and deafness. Arthrogryposis, dome-shaped skull, kyphosis, torticollis, lordosis, scoliosis, and spina bifida were the diagnosed skeletal defects. Upon necropsy, hydranencephaly, hydrocephaly, and microencephaly were seen in the calves presenting neurologic signs, while astrocytosis, astrogliosis, focal gliosis, perivascular, perineuronal, and submeningeal edema, perivascular cuffing, non-suppurative meningitis, non-suppurative encephalitis and lymphoplasmacytic infiltration, and perivascular and parenchymal hemorrhage were seen in samples obtained from the brains. RT-PCR detected Akabane virus in the brain tissues of the affected calves. This is the first clinical study of Akabane disease in calves in Iran.

Skeletal pathology in mouse models of Gould syndrome is partially alleviated by genetically reducing TGFβ signaling

Skeletal defects are hallmark features of many extracellular matrix (ECM) and collagen-related disorders. However, a biological function in bone has never been defined for the highly evolutionarily conserved type IV collagen. Collagen type IV alpha 1 (COL4A1) and alpha 2 (COL4A2) form α1α1α2 (IV) heterotrimers that represent a fundamental basement membrane constituent present in every organ of the body, including the skeleton. COL4A1 and COL4A2 mutations cause Gould syndrome, a variable and clinically heterogenous multisystem disorder generally characterized by the presence of cerebrovascular disease with ocular, renal, and muscular manifestations. We have previously identified elevated TGFβ signaling as a pathological insult resulting from Col4a1 mutations and demonstrated that reducing TGFβ signaling ameliorate ocular and cerebrovascular phenotypes in Col4a1 mutant mouse models of Gould syndrome. In this study, we describe the first characterization of skeletal defects in Col4a1 mutant mice that include a developmental delay in osteogenesis and structural, biomechanical and vascular alterations of mature bones. Using distinct mouse models, we show that allelic heterogeneity influences the presentation of skeletal pathology resulting from Col4a1 mutations. Importantly, we found that TGFβ target gene expression is elevated in developing bones from Col4a1 mutant mice and show that genetically reducing TGFβ signaling partially ameliorates skeletal manifestations. Collectively, these findings identify a novel and unsuspected role for type IV collagen in bone biology, expand the spectrum of manifestations associated with Gould syndrome to include skeletal abnormalities, and implicate elevated TGFβ signaling in skeletal pathogenesis in Col4a1 mutant mice.

A genetic mouse model mimicking MET related human osteofibrous dysplasia is characterized by delays in fracture repair and defective osteogenesis.

Osteofibrous dysplasia (OFD) is a rare, benign, fibro-osseous lesion that occurs most commonly in the tibia of children. Tibial involvement leads to bowing and predisposes to the development of a fracture which exhibit significantly delayed healing processes, leading to prolonged morbidity. We previously identified gain-of-function mutations in the MET gene as a cause for OFD. In our present study, we test the hypothesis that gain-of-function MET mutations impair bone repair due to reduced osteoblast differentiation. A heterozygous Met exon 15 skipping (MetΔ15-HET) mouse was created to imitate the human OFD mutation. The mutation results in aberrant and dysregulation of MET-related signaling determined by RNA-seq in the murine osteoblasts extracted from the wide-type and genetic mice. Although no gross skeletal defects were identified in the mice, fracture repair was delayed in MetΔ15-HET mice, with decreased bone formation observed 2-week postfracture. Our data are consistent with a novel role for MET-mediated signaling regulating osteogenesis.

Synergistic utilization of cost-effective glycerophosphate and biologically active zein for innovative minimally invasive smart thermo-responsive hydrogels for potential hard tissue engineering applications

Skeletal defects are the second-leading cause of disability worldwide, prompting the development of smart solutions for treatment. Calcium glycerophosphate (Ca–GP), chitosan (CS), hydroxyapatite (HA), and zein (ZN) were used to fabricate these thermo-responsive hydrogels. Ca–GP, an economically viable and bioactive glycerophosphate source, remains relatively underexplored. Natural protein ZN and the gold standard bone regenerative biomaterial HA were incorporated as reinforcing agents. The resulting composite hydrogels (HGs) exhibit a sol phase at 4 °C–10 °C and transition to gels at body temperature within 4–6 min. Their good injectability and the ability to be easily shaped into complex structures further support their great potential as minimally invasive solutions for treatment. The addition of ZN significantly improved the mechanical and biological properties of the HGs. The highest ZN concentration resulted in the strongest mechanical strength, measuring 52.2 MPa at 40% strain. HGs exhibited optimal swelling and degradation rates. Scanning electron microscopy analysis supported their porous nature. In vitro cell culture assays and wound healing assays demonstrated their excellent biocompatibility and regenerative potential. Drug-loaded HGs exhibited up to 90% drug release and antibacterial activity. All these results support their promising potential to support the regeneration of skeletal defects in a minimally invasive manner.

Variants in both the N- or C-terminal domains of IHH lead to defective secretion causing short stature and skeletal defects.

Heterozygous Indian Hedgehog gene (IHH) variants are associated with brachydactyly type A1 (BDA1). However, in recent years, numerous variants have been identified in patients with short stature and more variable forms of brachydactyly. Many are located in the C-terminal domain of IHH (IHH-C), which lacks signaling activity but is critical for auto-cleavage and activation of the N-terminal (IHH-N) peptide. The absence of functional studies of IHH variants, particularly for those located in IHH-C, has led to these variants being classified as variants of uncertain significance (VUS). To establish a simple functional assay to determine the pathogenicity of IHH VUS and confirm that variants in the C-terminal domain affect protein function. In vitro studies were performed for 9 IHH heterozygous variants, to test their effect on secretion and IHH intracellular processing by western blot of cells expressing each variant. IHH secretion was significantly reduced in all mutants, regardless of the location. Similarly, intracellular levels of N-terminal and C-terminal IHH peptides were severely reduced in comparison with the control. Two variants present at a relatively high frequency in the general population also reduced secretion but to a lesser degree in the heterozygous state. These studies provide the first evidence that variants in the C-terminal domain affect the secretion capacity of IHH and thus, reduce availability of IHH ligand, resulting in short stature and mild skeletal defects. The secretion assay permits a relatively easy test to determine the pathogenicity of IHH variants. All studied variants affected secretion and interestingly, more frequent population variants appear to have a deleterious effect and thus contribute to height variation.

Surface bioactivation of Polyetheretherketone (PEEK) by magnesium chondroitin sulfate (MgCS) as orthopedic implants for reconstruction of skeletal defects

Polyether-ether-ketone (PEEK) is clinically used as a bio-implant for the healing of skeletal defects. However, the osseointegration of clinical-sized bone grafts remains limited. In this study, surface-porous PEEK was created by using a sulfonation method and a metal-polysaccharide complex MgCS was introduced on the surface of sulfonated PEEK to form MgCS@SPEEK. The as-prepared MgCS@SPEEK was found to have a porous surface with good hydrophilicity and bioactivity. This was followed by an investigation into whether MgCS loaded onto sulfonated PEEK surfaces could promote osseointegration and angiogenesis. The in vitro results showed that MgCS@SPEEK had a positive effect on reducing the expression levels of inflammatory genes and promoting osteogenesis and angiogenesis-related genes expression levels. Furthermore, porous MgCS@SPEEK was implanted in critical-sized rat tibial defects for in vivo evaluation of osseointegration. The micro-computed tomography evaluation results revealed substantial bone formation at 4 and 8 weeks. Collectively, these findings indicate that MgCS@SPEEK could provide improved osseointegration and an attractive strategy for orthopaedic applications.