Mature Bone Research Articles

Vertical Alveolar Ridge Regeneration by Means of Periosteal Activation-A Proof-of-Principle Study.

To assess the possibility of vertical alveolar ridge augmentation by means of activation of the periosteum. Six adult male Beagle dogs were used for the study. All premolars and first molars were extracted, and one vertical saucer-shaped bony defect was created on each side of the mandible. After 3 months of healing, full-thickness muco-periosteal flaps were elevated, and one distraction device was placed on each side of the mandible. The distraction plate was left submerged, and the activation mechanism connected to the distraction rod was exposed intra-orally. The protocol of periosteal activation (PP: periosteal 'pumping') was initiated after a latency of 7 days. The alternation of activation and relaxation at the rate of 0.35 mm/12 h during 5 days was followed by the sole activation of 0.35 mm/12 h for 5 days (PP group). Devices were left inactivated on the contralateral control side of the mandible (C group). All animals were euthanized after 8 weeks of consolidation. Samples were analysed histologically and by means of micro-CT. New mature lamellar bone was formed over the pristine bone in all groups. More intensive signs of bone modelling and remodelling were observed in the PP group compared to the C group. Mean new bone, bone marrow, connective tissue and total volumetric densities were greater in the PP group (p < 0.001, p = 0.001, p = 0.003 and p < 0.001, respectively). No differences were observed in the relative area parameters. Total tissue volume and bone volume were higher in the PP group (p = 0.031 and p = 0.076, respectively), while the bone mineral densities were higher in the C group (p = 0.041 and p = 0.003, respectively). Trabecular number, trabecular thickness and trabecular separation values were similar between the two groups. Regeneration of vertical alveolar bone ridge defects may be enhanced by activation of the periosteum, without the application of bone grafting materials.

Hypergravity as a Possible Way of Bone Tissue Engineering in Osteoblastic Differentiation from Mesenchymal Stem Cells: A Systematic Review

Background: Tissue engineering development has become a highlight in recent decades. One of the key areas of focus is producing mature bone tissue to overcome orthopedic problems, such as bone defects. Various cultures have been implemented on stem cells; some induce osteoblastic differentiation markers, while others have the opposite effect. Microgravity has been proven in several studies to inhibit the expression of osteogenic differentiation markers. Conversely, hypergravity is expected to have the opposite impact, supporting stem cells in the osteogenesis pathway. Methods: A literature search was conducted using online databases including Sciencedirect, PubMed, and Proquest, covering the period from 2008 to 2022. This search considered only experimental studies published in English. The keywords used in this research were "hypergravity" and "mesenchymal stem cell." All acquired data were processed and analyzed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines (2020). Results: Initially, 190 studies were collected from online databases based on relevant keywords. After screening, 5 studies were included in the final analysis, focusing on hypergravity treatment and its effects on mesenchymal stem cells (MSCs). Conclusion: Hypergravity shows a significant and strong impact on osteoblastic differentiation. This study revealed that a gravity force of 30G and a culture duration of 7 to 14 days are the most optimal combination for inducing osteoblastic differentiation in MSCs.

DLK1 and DLK2, two non-canonical ligands of NOTCH receptors, differentially modulate the osteogenic differentiation of mesenchymal C3H10T1/2 cells

BackgroundC3H10T1/2 is a mesenchymal cell line capable of differentiating into osteoblasts, adipocytes and chondrocytes. The differentiation of these cells into osteoblasts is modulated by various transcription factors, such as RUNX2. Additionally, several interconnected signaling pathways, including the NOTCH pathway, play a crucial role in modulating their differentiation into mature bone cells. We have investigated the roles of DLK1 and DLK2, two non-canonical inhibitory ligands of NOTCH receptors, in the osteogenic differentiation of C3H10T1/2 cells.ResultsOur results corroborate existing evidence that DLK1 acts as an inhibitor of osteogenesis. In contrast, we demonstrate for the first time that DLK2 enhances this differentiation process. Additionally, our data suggest that NOTCH2, 3 and 4 receptors may promote osteogenesis, as indicated by their increased expression during this process, whereas NOTCH1 expression, which decreases during cell differentiation, might inhibit osteogenesis. Moreover, treatment with DAPT, a NOTCH signaling inhibitor, impeded osteogenic differentiation. We have confirmed the increase in ERK1/2 MAPK and p38 MAPK phosphorylation in C3H10T1/2 cells induced to differentiate to osteoblasts. Our new findings reveal increased ERK1/2 MAPK phosphorylation in differentiated C3H10T1/2 cells with a decrease in DLK1 expression or an overexpression of DLK2, which is coincident with the behavior of those transfectants where we have detected an increase in osteogenic differentiation. Additionally, p38 MAPK phosphorylation increases in differentiated C3H10T1/2 cells with reduced DLK1 levels.ConclusionsOur results suggest that DLK1 may inhibit osteogenesis, while DLK2 may promote it, by modulating NOTCH signaling and the phosphorylation of ERK1/2 and p38 MAPK pathways. Given the established inhibitory effect of DLK proteins on NOTCH signaling, these new insights could pave the way for developing future therapeutic strategies aimed at treating bone diseases.

Bone mineral density affects tumor growth by shaping microenvironmental heterogeneity

Breast cancer bone metastasis is a major cause of mortality in patients with advanced breast cancer. Although decreased mineral density is a known risk factor for bone metastasis, the underlying mechanisms remain poorly understood because studying the isolated effect of bone mineral density on tumor heterogeneity is challenging with conventional approaches. Moreover, mineralized biomaterials are commonly utilized for clinical bone defect repair, but how mineralized biomaterials affect the foreign body response and wound healing is unclear. Here, we investigate how bone mineral affects tumor growth and microenvironmental complexity in vivo by combining single-cell RNA-sequencing with mineral-containing or mineral-free decellularized bone matrices. We discover that the absence of bone mineral significantly influences fibroblast and immune cell heterogeneity, promoting phenotypes that increase tumor growth and alter the response to injury or disease. Importantly, we observe that the stromal response to bone mineral content depends on the murine tumor model used. While lack of bone mineral induces tumor-promoting microenvironments in both immunocompromised and immunocompetent animals, these changes are mediated by altered fibroblast phenotype in immunocompromised mice and macrophage polarization in immunocompetent mice. Collectively, our findings suggest that bone mineral density affects tumor growth by impacting microenvironmental complexity in an organism-dependent manner.

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.

WISH-BONE: Whole-mount insitu histology, to label osteocyte mRNA and protein in 3D adult mouse bones.

Bone is a three-dimensional (3D) highly dynamic tissue under constant remodeling. Commonly used tools to investigate bone biology require sample digestion for biomolecule extraction or provide only two-dimensional (2D) spatial information. There is a need for 3D tools to investigate spatially preserved biomarker expression in osteocytes. In this work, we present a new method, WISH-BONE, to label osteocyte messenger RNA (mRNA) and protein in whole-mount mouse bone. For mRNA labeling, we used hybridization chain reaction-fluorescence insitu hybridization (HCR-FISH) to label genes of interest in osteocytes. For protein labeling, samples were preserved using an epoxy-based solution that protects tissue structure and biomolecular components. Then an enzymatic matrix permeabilization step was performed to enable antibody penetration. Immunostaining was used to label various proteins involved in bone homeostasis. We also demonstrate the use of customized fluorescent nanobodies to target and label proteins in the cortical bone (CB). However, the relatively dim signal observed from nanobodies' staining limited detection. mRNA and protein labeling were performed in separate samples. In this study, we share protocols, highlight opportunities, and identify the challenges of this novel 3D labeling method. They are the first protocols for whole-mount osteocyte 3D labeling of mRNA and protein in mature mouse bones. WISH-BONE will allow the investigation of molecular signaling in bone cells in their 3D environment and could be applied to various bone-related fields of research.

The effect of radiotherapy, chemotherapy, and immunotherapy on fusion rate in spinal surgery using osteobiologics for patients with metastatic spinal disease: a systematic review.

To evaluate the impact that adjuvant therapies like radiotherapy, chemotherapy, and immunotherapy have on osteobiologic properties and bony regeneration in patients with metastatic spine disease (MSD) undergoing spinal fusion surgery. PubMed and ClinicalTrials.gov searches were performed. MSD patients undergoing fusion surgery with an osteobiologic and radiotherapy, chemotherapy and/or immunotherapy were included. Demographics, primary tumor, surgery, adjuvant treatments, osteobiologic type, fusion rates with scoring criteria, hardware failure, reoperation rates, follow-up, and survival were extracted. 1487 studies were screened, 20 included. 585 patients (464 with MSD) had fusion rates ranging from 17.9 to 100%. In the setting of radiotherapy, fusion rates of 10 studies using autologous bone graft (autograft), 5 studies using allogenic bone graft (allograft), 5 studies using combination autograft/allograft, 4 studies using biomaterial scaffolds (BMS), 3 studies using demineralized bone matrices (DBM), and 1 study using growth factors (GF), were 50-100%, 17.9-100%, 57.8-100%, 52.9-100%, 20-100%, and 100%, respectively. A higher incidence of fusion in patients with autograft or allograft receiving stereotactic body radiotherapy (SBRT) at lower biologically effective doses (BED) and at least 1-month postoperatively was noted. Chemotherapy had no impact on fusion. No studies evaluated the impact of immunotherapy on fusion. SBRT at lower doses given greater than 1-month postoperatively may enhance bony fusion in patients receiving autograft, allograft, or autograft/allograft. Chemotherapy may delay bony fusion without affecting overall fusion rates. Preclinical studies suggest immunotherapy may prevent osteolysis and promote osteogenesis, but no studies have yet evaluated the clinical impact of these findings on spinal fusion. Further research is needed on osteobiologics in bony regeneration in the MSD population.

Total body irradiation is associated with long-term deficits in femoral bone structure but not mechanical properties in male rhesus macaques.

Exposure to ionizing radiation for oncological therapy increases the risk for late-onset fractures in survivors. However, the effects of total body irradiation (TBI) on adult bone are not well-characterized. The primary aim of this study was to quantify the long-term effects of TBI on bone microstructure, material composition, and mechanical behavior in skeletally mature rhesus macaque (Macaca mulatta) non-human primates. Femora were obtained post-mortem from animals exposed to an acute dose of TBI (6.0-6.75Gy) nearly a decade earlier, age-matched non-irradiated controls, and non-irradiated young animals. The microstructure of femoral trabecular and cortical bone was assessed via micro-computed tomography. Material composition was evaluated by measuring total fluorescent advanced glycation end products (fAGEs). Cortical bone mechanical behavior was quantified via four-point bending and cyclic reference point indentation (cRPI). Animals exposed to TBI had slightly worse cortical microstructure, including lower cortical thickness (-11%, p = 0.037) and cortical area (-24%, p = 0.049), but similar fAGE content and mechanical properties as age-matched controls. Aging did not influence cortical microstructure, fAGE content, or cRPI measures but diminished femoral cortical post-yield properties, including toughness to fracture (-32%, p = 0.032). Because TBI was administered after the acquisition of peak bone mass, these results suggest that the skeletons of long-term survivors of adulthood TBI may be resilient, retaining or recovering their mechanical integrity during the post-treatment period, despite radiation-induced architectural deficits. Further investigation is necessary to better understand radiation-induced skeletal fragility in mature and immature bone to improve care for radiation patients of all ages.

8141 A Rare Case of Follicular Adenoma with Osseous Metaplasia and Mature Bone Formation- Is Surveillance Important?

Abstract Disclosure: K. Madani: None. Y. Al-Khazraji: None. D.H. Sacoto: None. G.K. Rai: None. R. Belokovskaya: None. A.A. Franco-Akel, MD: None. Osseous metaplasia and mature bone formation (MBF) are common in malignant thyroid and parathyroid tumors. It is very unusual to have MBF in a benign tumor-like follicular adenoma, as in the case we describe. We found only twelve such cases reported in the literature, and none addressed the cancer risk or need for surveillance for the development of potential malignancy. Thus, further studies are required to investigate the need for surveillance in patients not undergoing excision and in those with remnant thyroid tissue post-removal of the adenoma for recurrence. A 43-year-old female with history of hyperlipidemia and hypothyroidism had an incidental finding of midline neck mass during the evaluation of a left upper extremity lipoma. Ultrasound of the neck showed two nodules in the Left (L) thyroid lobe: a complex nodule measured 2.2 x 1.2 x 2.1 cm and a calcified nodule measuring 1.5 x 0.8 x 0.6 cm. MRI of the neck revealed a 1.9 cm heterogeneously enhancing nodule in the mid to lower pole of the L thyroid lobe. The patient had symptoms suggestive of compression, such as dysphagia, odynophagia, and hoarseness; thus, left hemothyroidectomy was performed. Tissue biopsy was notable for follicular adenoma with osseous metaplasia and mature bone formation. The patient remained clinically and biochemically euthyroid, with only mildly elevated thyrotropin level, and treatment with levothyroxine 25 mcg once daily was initiated. On post-surgical follow-up, there was a lack of data to support close monitoring modality and strategy for the remnant thyroid lobe and neck nodal compartments. We concluded with the patient to follow up in a clinical visit for a physical exam of the neck and to obtain a follow-up sonogram at an interval of six months, in addition to monitoring TSH levels to guide hypothyroidism therapy. Osseous metaplasia has not been established as a premalignant condition, but in certain tissues like the esophagus, metaplasia is a risk factor for malignant conversion, for instance, in Barrett’s esophagus leading to esophageal adenocarcinoma. Moreover, MBF in thyroid tissue has been attributed to the activity of basic fibroblast growth factor (bFGF) and bone morphogenetic protein-2 (BMP-2), which are linked with mitogenesis and tumorigenesis. Thus, the question arises if post-excision surveillance is required in patients with a history of osseous metaplasia in remnant thyroid tissue post-lobectomy or partial thyroidectomy. At this point, more epidemiological data on similar cases may help us understand the true risk of this clinical scenario, in order to develop a reasonable approach for surveillance. Presentation: 6/3/2024

A-050 Reference Intervals for Plasma Concentrations of C-Terminal Telopeptide of Type-I Collagen (CTX) for Greek Adult Population

Abstract Background CTX assays measure a fragment of the C-terminal telopeptide of type I collagen released during resorption of mature bone. To adequately interpret CTX results, method-specific and population specific reference intervals are needed. We aimed to determine reference intervals for plasma concentrations of CTX. Methods We collected samples from 431 apparently healthy Greeks (140 men as well as 150 pre- and 139 postmenopausal women), who volunteered to participate in our study. Data on socio-demographic characteristics, medical histories and medications were collected and subjects with conditions or receiving medications that affecting bone metabolism were excluded. Dual-energy x-ray absorptiometry (DXA) used to measure bone mineral density (BMD) in all participants. All blood collections were performed in the morning after overnight fast. Plasma EDTA samples were used for the measurement of CTX. Measurements were performed by an automated chemiluminescent immunoassay on the Maglumi-X3 analyzer (Snibe, Shenzhen, China). The reference interval was defined as the central 95% range and determined according to CLSI guide C28-A3 using the MedCalc Software. Results The table summarizes our results. The mean age of men, pre- and post-menopausal women were 50.7, 40,7 and 58.4 years respectively. DXA results revealed that 348 participants had normal BMD, 74 HAD osteopenia and 9 had osteoporosis which excluded from analysis. Since our data were not normally distributed (Shapiro-Wilk test) in any group and after the exclusion of outliers (Tukey test), we used the non-parametric method suggested by the CLSI guide to determine the reference intervals. Multiple regression analysis also revealed that sex menopausal status and BMD were factors affecting CTX values. Conclusions We provide reference intervals for the determination of CTX concentrations in EDTA-plasma for the Greek adult population using an automated immunoassay. Our data may aid to interpret bone turnover in the Greek adult population.

Eagle syndrome: tissue characteristics and structure of the styloid process.

Eagle syndrome is a bone disease where elongation of the styloid process leads to throat and neck pain, and in severe cases neurovascular symptoms such as syncope and neuralgia. The pathophysiology of Eagle syndrome is poorly understood with various theories having been proposed how this elongation is caused. To better understand the pathophysiology, we performed a work-up in 6 patients presenting with Eagle syndrome. Patients mainly presented with pain on turning the neck (100%), foreign body sensation (67%), tension in the neck (67%), and dysphagia (50%). The typical length of the styloid process ranges from 25 to 30 mm; however, [18F]NaF (sodium fluoride) PET/CT showed elongated styloid processes with an average length of 52.1 ± 15.6mm (mean ± SD) with increased turnover at the base of one of the styloid processes. The removed styloid processes were further examined by histology, micro-CT, quantitative backscatter electron imaging (qBEI), Fourier transform infrared spectroscopy (FTIR), and circularly polarized light imaging. Histology revealed one case of a fractured styloid process healing through callus formation and one case of pseudarthrosis. Bone mineral density and mineralization was similar in the styloid processes when compared to cortical bone samples derived from the mandibular bone of different patients. Circular polarized light microscopy showed a collagen orientation in the styloid process comparable to the cortical bone samples with a distinct separation of collagen structure between the mineralized structure and the surrounding soft tissue with FTIR analysis demonstrating a typical composition of bone. This altogether suggests that the elongated styloid processes in Eagle syndrome are mature bone, capable of endochondral repair, possibly growing from the base of the process through endochondral ossification, rather than being a form of secondary calcification of the stylohyoid ligament as previously postulated.

Profound deficiencies in mature blood and bone marrow progenitor dendritic cells in Chronic Lymphocyticcytic Leukemia patients.

Chronic lymphocytic leukemia (CLL) patients are immunocompromised and highly vulnerable to serious recurrent infections. Conventional dendritic cells (cDCs) and plasmacytoid DCs (pDCs) are principal sensors of infection and are essential in orchestrating innate and adaptive immune responses to resolve infection. This study identified significant deficiencies in six functionally distinct DC subsets in blood of untreated CLL (UT-CLL) patients and selective normalization of pDCs in response to acalabrutinib (a Bruton tyrosine kinase inhibitor) therapy. DCs are continuously replenished from hematopoiesis in bone marrow (BM). Four BM developmental intermediates that give rise to cDCs and pDCs were examined and significant reductions of these were identified in UT-CLL patients supporting a precursor/progeny relationship. The deficiencies in blood DCs and BM DC progenitors were significantly associated with alterations in the Flt3/FL signaling pathway critical to DC development and function. Regarding clinical parameter, cDC subset deficiencies are associated with adverse prognostic indicators of disease progression, including IGHV mutation, CD49d, CD38, and ZAP-70 status. Importantly, UT-CLL patients with shared DC subset deficiencies had shorter time-to-first treatment (TTFT), uncovering a profound alteration in innate immunity with the potential to instruct therapeutic decision-making.

Evaluation of biocompatibility and osseointegration of multi-component TiAl6V4 titanium alloy implants.

This study aimed to investigate the biocompatibility and osseointegration of novel titanium (Ti) implants with a perforated part with high surface roughness (Ra >4 μm) and a smooth solid part (test group), as compared to smooth solid Ti implants (control group; Ra < 0.8 μm). Test and control implants were implanted in rabbit femurs. After 4 and 15 weeks, host tissue reaction and quality of tissue formed were evaluated with histopathology, while micro-CT scans were used to quantitatively assess bone-implant contact (BIC), surrounding bone formation, and bone ingrowth. After 4 and 15 weeks, minimal host reaction was found in the test group. Histopathological analysis showed new bone formation around the implants in both the test and control groups after 4 weeks. Furthermore, additional bone growth was often observed within the holes of the test implants. After 15 weeks, the test implants showed high bone ingrowth and the presence of mature bone in direct contact with the implant surface, whereas, bone ingrowth was poorer for the control group with 30% of the control implants, showing larger gaps at the bone-implant interface. Quantitative micro-CT analysis revealed comparable BIC and bone formation in both groups at 4 weeks, but higher BIC and more bone formation in the test group than in the control group after 15 weeks. No significant differences were observed in any of the analyses. In conclusion, partially perforated, high-roughness Ti implants showed excellent osseointegration and minimal host reaction, indicating their potential for orthopedic applications in bone repair and regeneration.

Design of functionally graded porous lattice structure tibial implant for TAR

Use of porous lattice structures and functionally graded (FG) implants in orthopaedic applications has emerged as a promising solution to reduce stress shielding. However, there is a notable research gap in examining the effects of post-operative scenarios within the lattice pores and different porosity distribution laws in functionally graded porous lattice structure (FGPLS) implants on the biomechanical performance of tibial implant for total ankle replacement (TAR). The objective of the study is to investigates the effects of both post-operative tissues and various porosity distribution laws on the biomechanical performance of tibial implant for TAR and proposes a new design strategy. CAD models for lattice structures at various porosities were developed to incorporate the effects of post-operative tissues such as fibrous tissue, cartilage, immature bone, and mature bone. The pore spaces of these lattice structures were assumed to be filled with the post-operative ingrowth tissues. The lattice structure and these tissues together form the Representative volume element (RVE), whose effective properties were calculated using Asymptotic homogenization (AH) technique. Equations linking mechanical properties and porosity were established and used to assign mechanical properties to macro-FE models of FGPLS tibial implants based on three different power laws i.e. n = 0.1,1 and 5. Macro-FE models for intact and implanted tibia bones were developed using homogeneous properties of cortical bone and heterogeneous cancellous bone properties. The proximal part of the tibia was fixed, and a compressive load was applied through the anterior nodes of the meniscal bearing to represent dorsiflexion loading during normal walking. The Finite Element Analysis (FEA) was utilized to investigate the biomechanical performance of these implanted models in terms of stress distribution in the tibia bone and implant-bone micromotion. A comparison was made between models with solid metallic implants and those with FGPLS implants. Additionally, models with varying post-operative tissues in the pore space were compared, as well as models with different porosity variation laws were also compared. Results demonstrated that models implanted with FGPLS implants having porosity variation based on power law n = 0.1 showed increased bone stress and micromotion compared to those with solid metallic implants. Specifically, in models with FGPLS implants with porosity variation based on a power law of n = 0.1, the bone area with stress ranging from 2–5 MPa increased significantly in the periprosthetic region compared to models with solid metallic implants. The bone stress value for the majority of the region above the medial and lateral peg increased from 1–2 MPa to 2–5 MPa. Although there was a slight increase in micromotion values, they remained below the acceptable threshold of 50 µm. Models with porosity variations based on power laws n = 1 and n = 5 exhibited similar bone stress and micromotion results as solid metallic implants. Additionally, no significant difference in bone stresses was observed for different stages of bone ingrowth at pore space. These findings indicate that both the porous structure and porosity distribution within the implant significantly influence the biomechanical performance of tibial implants for TAR. So, it can be concluded that FGPLS implants with power law n = 0.1 are effective in reducing stress shielding and supporting the long-term survival of TAR by increasing bone stress and reducing the chances of aseptic loosening due to implant induced adaptive bone remodelling.

Development of a Biodegradable PLGA Carrier to Provide Wnt Agonists and Antibiotics to Meet the Requirements for Patients with Bone Infections.

Antibiotic beads can be used to treat surgical infections. In this study, polylactide-polyglycolide (PLGA) was mixed with vancomycin, the osteogenic enhancer lithium chloride (LiCl), and hot compression to form PLGA-vancomycin-LiCl delivery beads to treat bone infection. An elution method was used to characterize in vitro release characteristics of vancomycin and Li over a 42-day period. The release profiles lasted for more than 42 days for vancomycin and 28 days for Li. The concentration of vancomycin in each sample was well above the breakpoint sensitivity. Lithium cotreatment enhanced the bactericidal effect of vancomycin. Released Li and vancomycin increased the mRNA or protein expressions of osteogenic markers of mesenchymal stem cells (MSCs). In vivo, the PLGA delivery systems were implanted into the distal femoral cavities of rabbits, and the cavity fluid content was aspirated and analyzed at each time point. The released Li and vancomycin lasted more than 6 weeks, and the vancomycin concentrations were much greater than the breakpoint sensitivity. Four rabbits in each group were sacrificed at 8 weeks for histological observation. More mature bone tissue was observed in the Li treatment group. This study provides a PLGA drug delivery system to meet the requirements of patients with bone infections.

Quality assessment of regenerated bone in intraosseous and intramuscular scaffolds by spectroscopy and nanoindentation

The efficiency of synthetic bone grafts can be evaluated either in osseous sites, to analyze osteoconduction or ectopically, in intramuscular or subcutaneous sites, to assess osteoinduction. Bone regeneration is usually evaluated in terms of the presence and quantity of newly formed bone, but little information is normally provided on the quality of this bone. Here, we propose a novel approach to evaluate bone quality by the combined use of spectroscopy techniques and nanoindentation. Calcium phosphate scaffolds with different architectures, either foamed or 3D-printed, that were implanted in osseous or intramuscular defects in Beagle dogs for 6 or 12 weeks were analyzed. ATR-FTIR and Raman spectroscopy were performed, and mineral-to-matrix ratio, crystallinity, and mineral and collagen maturity were calculated and mapped for the newly regenerated bone and the mature cortical bone from the same specimen. For all the parameters studied, the newly-formed bone showed lower values than the mature host bone. Hardness and elastic modulus were determined by nanoindentation and, in line with what was observed by spectroscopy, lower values were observed in the regenerated bone than in the cortical bone. While, as expected, all techniques pointed to an increase in the maturity of the newly-formed bone between 6 and 12 weeks, the bone found in the intramuscular samples after 12 weeks presented lower mineralization than the intraosseous counterparts. Moreover, scaffold architecture also played a role in bone maturity, with the foamed scaffolds showing higher mineralization and crystallinity than the 3D-printed scaffolds after 12 weeks.

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.

The true history of the Hueter-Volkmann law.

The Hueter-Volkmann law (HVL) of the response of growth plate to compression load is a basic concept in orthopaedics. However, little is known about the origin of HVL and its history. A literature search was performed in original publications and historical sources. An analysis of all Volkmann´s and Hueter´s texts has shown that none of their publications was based on experiments, but on the data in the literature and their own clinical observations. They did not deal at all with the effect of pressure on the growth plate and mentioned this structure only marginally. The authors coined the opinion that increased pressure retards and decreased pressure accelerates bone growth. Julius Wolff criticized the HVL and concentrated all his arguments in the book "The law of bone remodeling". According to him, increased pressure leads to bone formation, decreased pressure to its resorption. The Wolff-Volkmann dispute was addressed in the German literature by a number of authors. Walther Müller in his monograph "The normal and pathological physiology of the bone" criticized Wolff for his concept of interstitial bone growth. In Müller´s view, HVL applies to the growing bone and Wolff confuses growth with hypertrophy of the mature bone. The circumstances of the emergence of HVL are inaccurately and incompletely described in the current literature, as they are mostly taken from secondary sources. HVL, as it is presented today, is not the original formulation, but the result of a long historical evolution.

Driving Osteocytogenesis from Mesenchymal Stem Cells in Osteon-like Biomimetic Nanofibrous Scaffolds.

The treatment of critical-sized bone defects caused by tumor removal, skeletal injuries, or infections continues to pose a major clinical challenge. A popular potential alternative solution to autologous bone grafts is a tissue-engineered approach that utilizes the combination of mesenchymal stromal/stem cells (MSCs) with synthetic biomaterial scaffolds. This approach aims to support new bone formation by mimicking many of the biochemical and biophysical cues present within native bone. Regrettably, osteocyte cells, crucial for bone maturation and homeostasis, are rarely produced within MSC-seeded scaffolds, thereby restricting the development of fully mature cortical bone from these synthetic implants. In this work, we have constructed a multimodal scaffold by combining electrospun poly(lactic-co-glycolic acid) (PLGA) fibrous scaffolds with poly(ethylene glycol) (PEG)-based hydrogels that mimic the functional unit of cortical bone, osteon (osteon-mimetic) scaffolds. These scaffolds were decorated with a novel bone morphogenic protein-6 (BMP6) peptide (BMP6p) after our findings revealed that the BMP6p drives higher levels of Smad signaling than the full-length protein counterpart, soluble or when bound to the PEG hydrogel backbone. We show that our osteon-mimetic scaffolds, in presenting concentric layers of BMP6p-PEG hydrogel overlaid on MSC-seeded PLGA nanofibers, promoted the rapid formation of osteocyte-like cells with a phenotypic dendritic morphology, producing early osteocyte markers, including E11/gp38 (E11). Maturation of these osteocyte-like cells was further confirmed by the observation of significant dentin matrix protein 1 (DMP1) throughout our bilayered scaffolds after 3 weeks, even when cultured in a medium without dexamethasone (DEX) or any other osteogenic supplements. These results demonstrate that these osteon-mimetic scaffolds, in presenting biochemical and topographical cues reminiscent of the forming osteon, can drive the formation of osteocyte-like cells in vitro from hBMSCs without the need for any osteogenic factor media supplementation.

Bone mineral density affects tumor growth by shaping microenvironmental heterogeneity.

Breast cancer bone metastasis is the leading cause of mortality in patients with advanced breast cancer. Although decreased mineral density is a known risk factor for bone metastasis, the underlying mechanisms remain poorly understood because studying the isolated effect of bone mineral density on tumor heterogeneity is challenging with conventional approaches. Here, we investigate how bone mineral content affects tumor growth and microenvironmental complexity in vivo by combining single-cell RNA-sequencing with mineral-containing or mineral-free decellularized bone matrices. We discover that the absence of bone mineral significantly influences fibroblast and immune cell heterogeneity, promoting phenotypes that increase tumor growth and alter the response to injury or disease. Importantly, we observe that the stromal response to matrix mineral content depends on host immunocompetence and the murine tumor model used. Collectively, our findings suggest that bone mineral density affects tumor growth by altering microenvironmental complexity in an organism-dependent manner.