Components of the Gut Microbiome That Influence Bone Tissue-Level Strength

Modifications to the constituents of the gut microbiome influence bone density and tissue-level strength, but the specific microbial components that influence tissue-level strength in bone are not known. Here, we selectively modify constituents of the gut microbiota using narrow-spectrum antibiotics to identify components of the microbiome associated with changes in bone mechanical and material properties. Male C57BL/6J mice (4 weeks) were divided into seven groups (n = 7–10/group) and had taxa within the gut microbiome removed through dosing with: (i) ampicillin; (ii) neomycin; (iii) vancomycin; (iv) metronidazole; (v) a cocktail of all four antibiotics together (with zero-calorie sweetener to ensure intake); (vi) zero-calorie sweetener only; or (vii) no additive (untreated) for 12 weeks. Individual antibiotics remove only some taxa from the gut, while the cocktail of all four removes almost all microbes. After accounting for differences in geometry, whole bone strength was reduced in animals with gut microbiome modified by neomycin (−28%, p = 0.002) and was increased in the group in which the gut microbiome was altered by sweetener alone (+39%, p < 0.001). Analysis of the fecal microbiota detected seven lower-ranked taxa differentially abundant in animals with impaired tissue-level strength and 14 differentially abundant taxa associated with increased tissue-level strength. Histological and serum markers of bone turnover and trabecular bone volume per tissue volume (BV/TV) did not differ among groups. These findings demonstrate that modifications to the taxonomic components of the gut microbiome have the potential to decrease or increase tissue-level strength of bone independent of bone quantity and without noticeable changes in bone turnover. © 2021 American Society for Bone and Mineral Research (ASBMR).     

Molecular and Cellular Mechanisms of Delayed Fracture Healing in Mmp10 (Stromelysin 2) Knockout Mice

The remodeling of the extracellular matrix is a central function in endochondral ossification and bone homeostasis. During secondary fracture healing, vascular invasion and bone growth requires the removal of the cartilage intermediate and the coordinate action of the collagenase matrix metalloproteinase (MMP)-13, produced by hypertrophic chondrocytes, and the gelatinase MMP-9, produced by cells of hematopoietic lineage. Interfering with these MMP activities results in impaired fracture healing characterized by cartilage accumulation and delayed vascularization. MMP-10, Stromelysin 2, a matrix metalloproteinase with high homology to MMP-3 (Stromelysin 1), presents a wide range of putative substrates identified in vitro, but its targets and functions in vivo and especially during fracture healing and bone homeostasis are not well defined. Here, we investigated the role of MMP-10 through bone regeneration in C57BL/6 mice. During secondary fracture healing, MMP-10 is expressed by hematopoietic cells and its maximum expression peak is associated with cartilage resorption at 14 days post fracture (dpf). In accordance with this expression pattern, when Mmp10 is globally silenced, we observed an impaired fracture-healing phenotype at 14 dpf, characterized by delayed cartilage resorption and TRAP-positive cell accumulation. This phenotype can be rescued by a non-competitive transplant of wild-type bone marrow, indicating that MMP-10 functions are required only in cells of hematopoietic linage. In addition, we found that this phenotype is a consequence of reduced gelatinase activity and the lack of proMMP-9 processing in macrophages. Our data provide evidence of the in vivo function of MMP-10 during endochondral ossification and defines the macrophages as the lead cell population in cartilage removal and vascular invasion. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).     

Preliminary Investigation of Airgap Electrospun Silk-Fibroin-Based Structures for Ligament Analogue Engineering

The process of electrospinning has proven to be highly beneficial for use in a number of tissue-engineering applications due to its ease of use, flexibility and tailorable properties. There have been many publications on the creation of aligned fibrous structures created through various forms of electrospinning, most involving the use of a metal target rotating at high speeds. This work focuses on the use of a variation known as airgap electrospinning, which does not use a metal collecting target but rather a pair of grounded electrodes equidistant from the charged polymer solution to create highly aligned 3D structures. This study involved a preliminary investigation and comparison of traditionally and airgap electrospun silk-fibroin-based ligament constructs. Structures were characterized with SEM and alignment FFT, and underwent porosity, permeability, and mechanical anisotropy evaluation. Preliminary cell culture with human dermal fibroblasts was performed to determine the degree of cellular orientation and penetration. Results showed airgap electrospun structures to be anisotropic with significantly increased porosity and cellular penetration compared to their traditionally electrospun counterparts.     

Adsorption of matrix metalloproteinases onto biomedical polymers: a new aspect in biological acceptance

Matrix metalloproteinases (MMPs) are zinc-dependent enzymes involved in the remodelling of connective tissues during the development and wound healing. Moreover, two MMPs, Gelatinase A (MMP-2) and Gelatinase B (MMP-9), are also present in body fluids such as blood and urine and, therefore, they can be in contact with implanted biomaterials and can be adsorbed onto their surface. In order to test this hypothesis disks of different polymers (polystyrene (PS), polyvinyl chloride (PVC), poly(D,L-lactide) (PLA), polymethyl methacrylate (PMMA) and poly(2-hydroxyethyl methacrylate) (PHEMA)) have been exposed to human plasma and adsorbed proteins have been eluted and analyzed. Using Western blot and substrate zymography analysis, we observed that both MMP-2 and MMP-9 adsorbed onto the surfaces of all the polymers, especially hydrophilic ones (PMMA and PHEMA) and PLA, in both the active and inactive forms. Furthermore, we observed that adhesion of human granulocyte neutophils to PMMA, the polymer that adsorbed the higher quantity of MMP-2 and MMP-9 compared to the others, was reduced by more that 50% by the presence of a gelatinase inhibitor. This data suggest a surprising role of these absorbed enzymes in the adhesion of neutrophil onto some polymeric biomaterials surface and, therefore, in the setting of inflammation.     

Extracellular and Intracellular Regulation of Biliary Lecithin Hydrophobicity

Bromosulfophthalein and papaverine have beendemonstrated to inhibit biliary lipid secretion withoutaffecting secretion of bile salts in normal rats,so-called uncoupling. Bromosulfophthalein inhibits the capacity of intracanalicular bile saltmicelles to induce biliary lipid secretion, andpapaverine inhibits vesicular transport within thehepatocyte. We compared the effects ofbromosulfophthalein and papaverine on biliary lipid secretion in normalSprague-Dawley rats and Eizai hyperbilirubinuria rats.The fatty acyl chain saturation in biliary lecithinincreased during bromosulfophthalein infusion and decreased during papaverine infusion inSprague-Dawley rats. Bromosulfophthalein had no effecton biliary lipid secretion in Eizai rats, whilepapaverine induced uncoupling. The degree of fatty acylchain saturation in biliary lecithin was unchangedduring bromosulfophthalein infusion, but decreased withpapaverine in Eizai rats. We deduce that selection ofbiliary lecithin species occurs at various points in the lipid transport pathway at intracellularand intracanalicular sites.     

Poly (N-isopropylacrylamide) (PNIPAM)-grafted gelatin as thermoresponsive three-dimensional artificial extracellular matrix: molecular and formulation parameters vs. cell proliferation potential

A series of poly(N-isopropylacrylamide)-grafted gelatins (PNIPAM gelatins) of three different graft densities (approx. 11, 22 and 34 graft chains per gelatin molecule) and three different molecular weights of their graft chains (molecular weight approximately 1.2 × 10⁴, 5.0 × 10⁴ and 1.3 × 10⁵ g/mol) were prepared by multiple derivatization of dithiocarbamyl (DC) group in a gelatin molecule and subsequent iniferter (acts as an initiator, transfer-agent and terminator)-based photopolymerization of NIPAM. The weight ratio of PNIPAM graft chains to gelatin (P/G) varied from 1.4 to 49. Aqueous solutions of PNIPAM-gelatins showed thermo-responsiveness, depended on the graft density and the molecular weight of PNIPAM graft chain or P/G. Aqueous solutions (10 or 20%, w/v) of PNIPAM-gelatins with P/G of more than 5.8 were converted to gels at 37°C. Focal plane images of PNIPAM-gelatin gels by confocal laser scanning microscopy revealed that the size of hydrophobically clustered aggregates increased with P/G, whereas the space of microvoids decreased with concentration. Compressive strain–stress measurements revealed that compressive strength of PNIPAM-gelatin increased with P/G. Bovine smooth muscle cells (SMCs)-entrapped gels were produced from PNIPAM-gelatin-containing cell-suspended medium solutions at 37°C. The entrapped cells proliferated in the gel with P/G of more than 12. A higher cell proliferativity was obtained at low concentration (5%, w/v) and higher P/G (> 18). Tissue formation composed of proliferative SMCs and cell-secreted extracellular matrices (collagen) was obtained at 14 days incubation. The inter-relationship between the molecular parameters of PNIPAM-gelatin, internal structural features and cell proliferation potential was discussed.     

Effects of Chitosan-Coated Fibers as a Scaffold for Three-Dimensional Cultures of Rabbit Fibroblasts for Ligament Tissue Engineering

Material selection in tissue-engineering scaffolds is one of the primary factors defining cellular response and matrix formation. In this study, we fabricated chitosan-coated poly(lactic acid) (PLA) fiber scaffolds to test our hypothesis that PLA fibers coated with chitosan highly promoted cell supporting properties compared to those without chitosan. Both PLA fibers (PLA group) and chitosan-coated PLA fibers (PLA–chitosan group) were fabricated for this study. Anterior cruciate ligament (ACL) fibroblasts were isolated from Japanese white rabbits and cultured on scaffolds consisting of each type of fiber. The effects of cell adhesivity, proliferation, and synthesis of the extracellular matrix (ECM) for each fiber were analyzed by cell counting, hydroxyproline assay, scanning electron microscopy and quantitative RT-PCR. Cell adhesivity, proliferation, hydroxyproline content and the expression of type-I collagen mRNA were significantly higher in the PLA–chitosan group than in the PLA group. Scanning electron microscopic observation showed that fibroblasts proliferated with a high level of ECM synthesis around the cells. Chitosan coating improved ACL fibroblast adhesion and proliferation, and had a positive effect on matrix production. Thus, the advantages of chitosan-coated PLA fibers show them to be a suitable biomaterial for ACL tissue-engineering scaffolds.     

An Investigation of the Mineral in Ductile and Brittle Cortical Mouse Bone

Bone is a strong and tough material composed of apatite mineral, organic matter, and water. Changes in composition and organization of these building blocks affect bone's mechanical integrity. Skeletal disorders often affect bone's mineral phase, either by variations in the collagen or directly altering mineralization. The aim of the current study was to explore the differences in the mineral of brittle and ductile cortical bone at the mineral (nm) and tissue (µm) levels using two mouse phenotypes. Osteogenesis imperfecta model, oim^‐/‐, mice have a defect in the collagen, which leads to brittle bone; PHOSPHO1 mutants, Phospho1^‐/‐, have ductile bone resulting from altered mineralization. Oim^‐/‐ and Phospho1^‐/‐ were compared with their respective wild‐type controls. Femora were defatted and ground to powder to measure average mineral crystal size using X‐ray diffraction (XRD) and to monitor the bulk mineral to matrix ratio via thermogravimetric analysis (TGA). XRD scans were run after TGA for phase identification to assess the fractions of hydroxyapatite and β‐tricalcium phosphate. Tibiae were embedded to measure elastic properties with nanoindentation and the extent of mineralization with backscattered electron microscopy (BSE SEM). Results revealed that although both pathology models had extremely different whole‐bone mechanics, they both had smaller apatite crystals, lower bulk mineral to matrix ratio, and showed more thermal conversion to β‐tricalcium phosphate than their wild types, indicating deviations from stoichiometric hydroxyapatite in the original mineral. In contrast, the degree of mineralization of bone matrix was different for each strain: brittle oim^‐/‐ were hypermineralized, whereas ductile Phospho1^‐/‐ were hypomineralized. Despite differences in the mineralization, nanoscale alterations in the mineral were associated with reduced tissue elastic moduli in both pathologies. Results indicated that alterations from normal crystal size, composition, and structure are correlated with reduced mechanical integrity of bone. © 2014 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.     

Decreased Mechanical Strength and Collagen Content in SPARC‐Null Periodontal Ligament Is Reversed by Inhibition of Transglutaminase Activity

The periodontal ligament (PDL) is a critical tissue that provides a physical link between the mineralized outer layer of the tooth and the alveolar bone. The PDL is composed primarily of nonmineralized fibrillar collagens. Expression of secreted protein acidic and rich in cysteine (SPARC/osteonectin), a collagen‐binding matricellular protein, has been shown to be essential for collagen homeostasis in PDL. In the absence of SPARC, PDL collagen fibers are smaller and less dense than fibers that constitute WT PDL. The aim of this study was to identify cellular mechanisms by which SPARC affected collagen fiber assembly and morphology in PDL. Cross‐linking of fibrillar collagens is one parameter that is known to affect insoluble collagen incorporation and fiber morphology. Herein, the reduction in collagen fiber size and quantity in the absence of SPARC expression was shown to result in a PDL with reduced molar extraction force in comparison to that of WT mice (C57Bl/6J). Furthermore, an increase in transglutaminase activity was found in SPARC‐null PDL by biochemical analyses that was supported by immunohistochemical results. Specifically, collagen I was identified as a substrate for transglutaminase in PDL and transglutaminase activity on collagen I was found to be greater in SPARC‐null tissues in comparison to WT. Strikingly, inhibition of transglutaminase activity in SPARC‐null PDL resulted in increases in both collagen fiber thickness and in collagen content, whereas transglutaminase inhibitors injected into WT mice resulted in increases in collagen fiber thickness only. Furthermore, PDL treated with transglutaminase inhibitors exhibited increases in molar extraction force in WT and in SPARC‐null mice. Thus, SPARC is proposed to act as a critical regulator of transglutaminase activity on collagen I with implications for mechanical strength of tissues. © 2015 American Society for Bone and Mineral Research