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Background: Extracellular vesicles (EVs) function as biomarkers and are crucial in cell communication and regulation, with therapeutic potential for intervertebral disc (IVD)-related low back pain (LBP). EV cargo is often affected by tissue health, which may affect the therapeutic potential. There is currently limited knowledge of how the cargo of IVD cell-derived EVs varies with tissue health and how differences in proteomic profile affect the predicted biological functions., Methods: Our study purified EVs from human IVD cell conditioned media by size-exclusion chromatography. Nanoparticle tracking analysis was conducted to measure EV size and concentration. Transmission electron microscopy and Western blot were performed to examine EV structure and markers. Tandem mass tag-mass spectrometry was conducted to determine protein cargo., Results: Most EVs were exosomes and intermediate microvesicles with an increasing amount linked to disease progression. Of the proteins detected, 88.6% were shared across the non-degenerate, mildly-degenerate, and degenerate samples. GO and KEGG analyses revealed that cargo from the mildly-degenerate samples was the most distinct, with the proteins in high abundance strongly associated with extracellular matrix (ECM) organization and structure. Shared proteins, highly expressed in the non-degenerate and degenerate samples, showed strong associations with cell adhesion, ECM-receptor interaction, and vesicle-mediated transport, respectively., Conclusions: Our findings indicate that EVs from IVD cells from tissue with different degrees of degeneration share a majority of the cargo proteins. However, the level of expression differs with degeneration grade. Cargo from the mildly-degenerate samples exhibits the most differences. A better understanding of changes in EV cargo in the degenerative process may provide novel information related to molecular mechanisms underlying IVD degeneration and suggest new potential treatment modalities for IVD-related LBP., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Author(s). JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Mesenchymal stem cells derived from adipose tissue have become a widely investigated cell source to use in tissue engineering applications. However, an optimal delivery scaffold for these cells is still needed. A rapidly gelling, injectable chitosan sponge was proposed in this study as a potential candidate for a suitable delivery scaffold. The results demonstrated the ability to encapsulate the stem cells at a 97.6% encapsulation efficiency and that the cells maintain their viability within the sponge. With the potential of using this scaffold for bone tissue engineering, ALP activity assay and fluorescent imaging for osteocalcin proved the ability to differentiate the encapsulated cells into the osteogenic lineage. Furthermore, co-encapsulation of pyrophosphatase within the sponge was investigated as a method to overcome the inhibitory effects that the sponge degradation by-products have on mineralization. Alizarin Red S staining demonstrated the beneficial effects of adding pyrophosphatase, where a significant increase in mineralization levels was achieved., (© 2021 Wiley Periodicals LLC.)

Pediatric patients with Osteogenesis Imperfecta (OI), a heritable connective tissue disorder, frequently suffer from long bone deformations. Surgical correction often results in bone non-unions, necessitating revision surgery with autogenous bone grafting using bone-marrow-derived stem cells (BM-SC) to regenerate bone. BM-SC harvest is generally invasive and limited in supply; thus, adipose tissue's stromal vascular fraction (SVF) has been introduced as an alternative stem cell reservoir. To elucidate if OI patients' surgical site dissected adipose tissue could be used as autologous bone graft in future, we investigated whether the underlying genetic condition alters SVF's cell populations and in vitro differentiation capacity. After optimizing SVF isolation, we demonstrate successful isolation of SVF of pediatric OI patients and non-OI controls. The number of viable cells was comparable between OI and controls, with about 450,000 per gram tissue. Age, sex, type of OI, disease-causing collagen mutation, or anatomical site of harvest did not affect cell outcome. Further, SVF-containing cell populations were similar between OI and controls, and all isolated SVF's demonstrated chondrogenic, adipogenic, and osteogenic differentiation capacity in vitro. These results indicate that SVF from pediatric OI patients could be used as a source of stem cells for autologous stem cell therapy in OI., (© 2022. The Author(s).)

Osteogenesis imperfecta (OI) type VI is a recessively inherited form of OI that is caused by mutations in SERPINF1, the gene coding for pigment-epithelium derived factor (PEDF). Here, we report on two apparently unrelated children with OI type VI who had the same unusual homozygous variant in intron 6 of SERPINF1 (c.787-10C>G). This variant created a novel splice site that led to the in-frame addition of three amino acids to PEDF (p.Lys262_Ile263insLeuSerGln). Western blotting showed that skin fibroblasts with this mutation produced PEDF but failed to secrete it. Both children were treated with intravenous bisphosphonates, but the treatment of Individual 1 was switched to subcutaneous injections of denosumab (dose 1 mg per kg body weight, repeated every 3 months). An iliac bone sample obtained after 5 denosumab injections (and 3 months after the last injection) showed no change in the increased osteoid parameters that are typical of OI type VI, but the number of osteoclasts in trabecular bone was markedly increased. This suggests that the effect of denosumab on osteoclast suppression is of shorter duration in children with OI type VI than what has previously been reported on adults with osteoporosis.

Osteogenesis imperfecta type VI is caused by mutations in SERPINF1, which codes for pigment-epithelium derived factor (PEDF). Most of the reported SERPINF1 mutations lead to premature termination codons, but three in-frame insertion or deletion mutations have also been reported. It is not clear how such in-frame mutations lead to OI type VI. In the present study we therefore investigated how SERPINF1 in-frame mutations affect the intracellular localization and secretion of PEDF. Skin fibroblasts affected by SERPINF1 in-frame mutations transcribed SERPINF1 at slightly reduced levels but secretion of PEDF was markedly diminished. Two deletions (p.F277del and the deletion of SERPINF1 exon 5) were associated with retention of PEDF in the endoplasmic reticulum and a stress response in osteoblastic cells. A recurrent in-frame duplication of three amino acids (p.Ala91_Ser93dup) appeared to lead to intracellular degradation but no retention in the endoplasmic reticulum or stress response. Immunofluorescence imaging in transiently transfected osteoblastic MC3T3-E1 cells suggested that PEDF affected by in-frame mutations was not transported along the secretory pathway. MC3T3-E1 osteoblasts stably overexpressing SERPINF1 with the p.Ala91_Ser93dup mutation had decreased collagen type I deposition and mineralization. Thus, the assessed homozygous in-frame deletions or insertions lead to retention or degradation within cellular compartments and thereby interfere with PEDF secretion., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Secreted protein, acidic, cysteine-rich (SPARC) is a glycoprotein that binds to collagen type I and other proteins in the extracellular matrix. Using whole-exome sequencing to identify the molecular defect in two unrelated girls with severe bone fragility and a clinical diagnosis of osteogenesis imperfecta type IV, we identified two homozygous variants in SPARC (GenBank: NM_003118.3; c.497G>A [p.Arg166His] in individual 1; c.787G>A [p.Glu263Lys] in individual 2). Published modeling and site-directed mutagenesis studies had previously shown that the residues substituted by these mutations form an intramolecular salt bridge in SPARC and are essential for the binding of SPARC to collagen type I. The amount of SPARC secreted by skin fibroblasts was reduced in individual 1 but appeared normal in individual 2. The migration of collagen type I alpha chains produced by these fibroblasts was mildly delayed on SDS-PAGE gel, suggesting some overmodification of collagen during triple helical formation. Pulse-chase experiments showed that collagen type I secretion was mildly delayed in skin fibroblasts from both individuals. Analysis of an iliac bone sample from individual 2 showed that trabecular bone was hypermineralized on the material level. In conclusion, these observations show that homozygous mutations in SPARC can give rise to severe bone fragility in humans., (Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

We had previously published the clinical characteristics of a bone fragility disorder in children that was characterized mainly by lower extremity fractures and a mineralization defect in bone tissue but not on the growth plate level. We have now performed whole-exome sequencing on four unrelated individuals with this phenotype. Three individuals were homozygous for a nucleotide change in BMP1, affecting the polyadenylation signal of the transcript that codes for the short isoform of BMP1 (BMP1-1) (c.*241T>C). In skin fibroblasts of these individuals, we found low levels of BMP1-1 transcript and protein. The fourth individual was compound heterozygous for the c.*241T>C variant in BMP1-1 and a variant in BMP1 exon 15 (c.2107G>C) that affected splicing in both BMP1-1 and the long isoform of BMP1 (BMP1-3). Both the homozygous 3'UTR variant and the compound heterozygous variants were associated with impaired procollagen type I C-propeptide cleavage, as the amount of free C-propeptide in the supernatant of skin fibroblasts was less than in controls. Peripheral quantitative computed tomography showed that all individuals had elevated volumetric cortical bone mineral density. Assessment of iliac bone samples by histomorphometry and quantitative backscattered electron imaging indicated that the onset of mineralization at bone formation sites was delayed, but that mineralized matrix was hypermineralized. These results show that isolated lack of BMP1-1 causes bone fragility in children., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Bone is one of the most dynamic tissues in the human body that can heal following injury without leaving a scar. However, in instances of extensive bone loss, this intrinsic capacity of bone to heal may not be sufficient and external intervention becomes necessary. Several techniques are available to address this problem, including autogenous bone grafts and allografts. However, all these techniques have their own limitations. An alternative method is the technique of distraction osteogenesis, where gradual and controlled distraction of two bony segments after osteotomy leads to induction of new bone formation. Although distraction osteogenesis usually gives satisfactory results, its major limitation is the prolonged duration of time required before the external fixator is removed, which may lead to numerous complications. Numerous methods to accelerate bone formation in the context of distraction osteogenesis have been reported. A viable alternative to autogenous bone grafts for a source of osteogenic cells is mesenchymal stem cells from bone marrow. However, there are certain problems with bone marrow aspirate. Hence, scientists have investigated other sources for mesenchymal stem cells, specifically adipose tissue, which has been shown to be an excellent source of mesenchymal stem cells. In this paper, the potential use of adipose stem cells to stimulate bone formation is discussed.

Context: Homozygous mutations in SERPINF1 cause deficiency of pigment epithelium-derived factor (PEDF) and lead to osteogenesis imperfecta (OI) type VI, but it is not known whether heterozygous mutations in SERPINF1 cause a phenotype., Objective: In the present study, we therefore assessed family members of individuals with OI type VI and compared the results of SERPINF1 mutation carriers with those of noncarriers of SERPINF1 mutations., Setting: This study was conducted at a metabolic bone clinic of a pediatric orthopedic hospital., Subjects: The study population comprised 29 family members (age range 8-89 y; 18 females, 11 males) of patients with a diagnosis of OI type VI. Eighteen individuals were heterozygous for SERPINF1 mutations, but the others did not carry a mutation., Main Outcome Measures: PEDF expression was assessed in skin fibroblasts from four heterozygous SERPINF1 mutation carriers. Skeletal characteristics and body composition were measured using dual-energy X-ray absorptiometry and peripheral quantitative computed tomography. Serum samples were used to quantify markers of bone metabolism, lipid status, and PEDF., Results: Carriers of heterozygous stop or frame shift mutations in SERPINF1 had low SERPINF1 transcript levels. Mean PEDF serum concentrations were significantly lower in the carrier group than in the noncarriers (P = .04). However, no group differences were found with regard to areal bone density at the lumbar spine and total body, volumetric bone density at the radius and tibia, body composition, lipid status, and markers of bone metabolism., Conclusion: Heterozygous SERPINF1 mutation carriers had no detectable abnormalities in fat and bone, despite decreased PEDF expression.

Recent reports have shown that homozygous or compound heterozygous mutations in WNT1 can give rise to severe bone fragility resembling osteogenesis imperfecta, whereas heterozygous WNT1 mutations have been found in adults with dominant early-onset osteoporosis. Here we assessed the effects of WNT1 mutations in four children with recessive severe bone fragility and in heterozygous family members. In vitro studies using the Topflash luciferase reporter system showed that two WNT1 missense mutations that were observed in these families, p.Cys143Phe and p.Val355Phe, decreased the ability of WNT1 to stimulate WNT signaling by >90%. Analyses of iliac bone samples revealed no major abnormalities in bone mineralization density distribution, an indicator of material bone properties, whereas a shift towards higher bone mineralization density is characteristic of classical osteogenesis imperfecta caused by mutations in COL1A1/COL1A2. Intravenous bisphosphonate treatment of four children with homozygous or compound heterozygous WNT1 mutations was associated with increasing lumbar spine areal bone mineral density z-scores, as measured by dual energy X-ray absorptiometry, but the effect was smaller than what had previously been reported for children with classical osteogenesis imperfecta. Family members with heterozygous WNT1 mutation tended to have low bone mass. Three of these heterozygous individuals had radiographic signs of vertebral fractures. These observations suggest that more effective treatment approaches are needed for children with recessive WNT1-related bone fragility and that a systematic work-up for osteoporosis is warranted for WNT1 mutation carriers in these families., (Copyright © 2014. Published by Elsevier Inc.)

Mutations in PLS3 have been identified as a cause of bone fragility in children, but the bone phenotype associated with PLS3 mutations has not been reported in detail. PLS3 is located on the X chromosome and encodes the actin-binding protein plastin 3. Here we describe skeletal findings in 4 boys from 2 families with mutations in PLS3 (c.994_995delGA; p.Asp332* in family 1; c.1433T > C; p.Leu478Pro in family 2). When first evaluated between 4 and 8 years of age, these boys had a history of one to four long-bone fractures. Mild vertebral compression fractures were identified in each boy. No obvious extraskeletal disease manifestations were present. Lumbar spine areal bone mineral density (LS-aBMD) Z-scores ranged from -1.7 to -3.5, but height was normal. Iliac bone histomorphometry in 2 patients showed low trabecular bone volume and a low osteoid maturation time but normal bone formation rate and osteoclast surface. Quantitative backscattered electron imaging (qBEI) did not reveal a major abnormality in bone mineralization density distribution. The 2 boys from family 1 received oral alendronate for 6 years, which normalized LS-aBMD. The mothers of the 4 boys did not have a history of fractures and had normal LS-aBMD. However, one of these mothers had low bone mass at the distal radius, as measured by peripheral quantitative computed tomography (pQCT). In conclusion, hemizygous mutations in PLS3 are associated with osteoporosis and bone fragility in childhood, but in contrast to bone fragility caused by mutations in collagen type I encoding genes, there is no hypermineralization of mineralized bone matrix., (© 2014 American Society for Bone and Mineral Research.)