Micro- and macromechanical characterization of the influence of surface-modification of poly(vinyl alcohol) fibers on the reinforcement of calcium phosphate cements.

Calcium phosphate cements (CPCs) are frequently used as synthetic bone substitute materials due to their favorable osteocompatibility and handling properties. However, CPCs alone are inherently brittle and exhibit low strength and toughness, which restricts their clinical applicability to non-load bearing sites. Mechanical reinforcement of CPCs using fibers has proven to be an effective strategy to toughen these cements by transferring stress from the matrix to the fibers through frictional sliding at the interface. Therefore, tailoring the fiber-matrix affinity is paramount in designing highly toughened CPCs. However, the mechanistic correlation between this interaction and the macromechanical properties of fiber-reinforced CPCs has hardly been investigated to date. The aim of this study was to tailor the fiber-matrix interface affinity by modifying the surface of poly(vinyl alcohol) (PVA) fibers and correlate their interfacial properties to macromechanical properties (i.e. fracture toughness, work-of-fracture and tensile strength) of CPCs. Results from single fiber pullout tests reveal that the surface modification of PVA fibers increased their hydrophilicity and improved their affinity to the CPC matrix. This observation was evidenced by an increase in the interfacial shear strength and a reduction in the critical fiber embedment length (i.e. maximum embedded length from which a fiber can be pulled out without rupture). This increased interface affinity facilitated energy dissipation during fracture of CPCs subjected to macromechanical three-point flexure and tensile tests. The fracture toughness also significantly improved, even for CPCs reinforced with fibers of lengths greater than their critical fiber embedment length, suggesting that other crack-arresting mechanisms also play an important role in mechanically reinforcing CPCs. Overall, these basic insights will improve the understanding of the correlation between micro- and macromechanical characteristics of fiber-reinforced CPCs.

Sterilization effects on the handling and degradation properties of calcium phosphate cements containing poly (D,L -lactic-co-glycolic acid) porogens and carboxymethyl cellulose.

Injectable, self-setting calcium phosphate cements (CPCs) are synthetic bone substitutes considered favorable for the repair and regeneration of bone due to their osteocompatibility and unique handling properties. However, their clinical applicability can be compromised due to insufficient cohesion upon injection into the body coupled with poor degradation rates that restricts new bone formation. Consequently, carboxymethyl cellulose (CMC) was incorporated into CPC formulations to improve their cohesion and injectability while poly (D,L -lactic-co-glycolic acid) (PLGA) porogens were added to introduce macroporosity and improve their biodegradation rate. Like most biomaterials, CPCs are gamma irradiated before clinical use to ensure sufficient sterilization. However, it is well known that gamma irradiation also reduces the molecular weight of CMC and PLGA via chain scission, which affects their material properties. Therefore, the aim of this study is to measure the effect that gamma irradiation has on the molecular weight of CMC at varying doses of 15, 40, or 80 kGy and investigate how this affects the handling (i.e., injectability, cohesion, washout, and setting times) and in vitro degradation behavior of CPC formulations. Results reveal that the molecular weight of CMC decreases with increasing gamma irradiation dose, thereby reducing the viscosifying capabilities of CMC, which causes CPCs to deteriorate more readily. Further, the addition of CMC seems to inhibit the degree of phase transformation during cement setting while the subsequent reduction in molecular weight of PLGA after gamma irradiation improves the in vitro degradation rate of CPCs due to the faster degradation rate of low molecular weight PLGA. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2216-2228, 2019.

Tough and Osteocompatible Calcium Phosphate Cements Reinforced with Poly(vinyl alcohol) Fibers.

Injectable, self-setting calcium phosphate cements (CPCs) are favorable bone substitutes due to their osteocompatibility. However, due to their brittleness and low toughness, their clinical application is limited to non-load-bearing sites. The incorporation of poly(vinyl alcohol) (PVA) fibers into cementitious materials is a successful strategy in civil engineering for improving the mechanical performance of cements. However, PVA fibers in particular have not yet been applied to reinforce CPCs. Therefore, the aim of this study is to investigate the effect of PVA fibers on the mechanical properties of CPCs. Second, the in vitro cytocompatibility of these fibers is studied using cell culture tests. Finally, the in vivo osteocompatibility of PVA fiber-reinforced CPCs is studied after a 6 and 12 week implantation period in the femoral condyle of rabbits. Results reveal that the incorporation of PVA fibers into CPCs is a highly effective strategy to strengthen and toughen CPCs, since the flexural strength and toughness of CPCs increased by more than 3-fold and 435-fold, respectively, upon reinforcement with PVA fibers. In vitro cytocompatibility tests indicate that PVA fibers are cytocompatible, which is further confirmed by the in vivo results that show that PVA fibers do not compromise the excellent osteocompatibility of CPCs.

Long-term biological performance of injectable and degradable calcium phosphate cement.

Enhancing degradation of poorly degrading injectable calcium phosphate (CaP) cements (CPCs) can be achieved by adding poly(lactic-co-glycolic acid) (PLGA) microparticles, generating porosity after polymer degradation. CPC-PLGA has proven to be biodegradable, although its long-term biological performance is still unknown. Optimization of injectability could be achieved via addition of carboxymethyl cellulose (CMC). Here, we evaluated the long-term in vivo performance of CPC-PLGA with or without the lubricant CMC in comparison to the devitalized bovine bone mineral (DBBM) predicate device Bio-Oss®. Rabbit femoral bone defects were injected with a CPC-formulation or filled with Bio-Oss® granules. Samples were retrieved at 6 and 26 weeks. Material degradation for Bio-Oss® was marginal, starting with 57% material remnants at implantation, 49% at 6 weeks, and 35% at 26 weeks, respectively. In contrast, CPC-PLGA and CPC-PLGA-CMC showed significant material degradation, starting with 100% material remnants at implantation, 56 and 78% at 6 weeks, and 8 and 21% at 26 weeks. Bone formation showed to be rapid for Bio-Oss®, with 24% at 6 weeks, and a similar value (27%) at 26 weeks. Both CPC-PLGA and CPC-PLGA-CMC showed a continuous temporal increase in bone formation, with 13 and 6% at 6 weeks, and 44 and 32% at 26 weeks. This study showed that CPC-PLGA induces favorable bone responses with >90% degradation and >40% new bone formation after an implantation period of 26 weeks.

Long-term evaluation of the degradation behavior of three apatite-forming calcium phosphate cements.

Calcium phosphate cements (CPCs) are injectable bone substitutes with a long clinical history because of their biocompatibility and osteoconductivity. Nevertheless, their cohesion upon injection into perfused bone defects as well as their long-term degradation behavior remain major clinical challenges. Therefore, the long-term degradation behavior of two types of α-tricalcium phosphate-based, apatite-forming CPCs was compared to a commercially available apatite-forming cement, that is HydroSet™ . Carboxyl methylcellulose (CMC) was used as cohesion promotor to improve handling properties of the two experimental cements, whereas poly (d, l-lactic-co-glycolic) acid (PLGA) microparticles were added to introduce macroporosity and stimulate CPC degradation. All three CPCs were injected into defects drilled into rabbit femoral condyles and explanted after 4, 12, or 26 weeks, after which the bone response was assessed both qualitatively and quantitatively. CPCs without PLGA microparticles degraded only at the periphery of the implants, while the residual CPC volume was close to 90%. On the contrary, bone ingrowth was observed not only at the periphery of the CPC, but also throughout the center of the implants after 26 weeks of implantation for the PLGA-containing CPCs with a residual CPC volume of approximately 55%. In conclusion, it was shown that CPC containing CMC and PLGA was able to induce partial degradation of apatite-forming CPCs and concomitant replacement by bone tissue.

Strain-guided mineralization in the bone-PDL-cementum complex of a rat periodontium.

OBJECTIVE: The objective of this study was to investigate the effect of mechanical strain by mapping physicochemical properties at periodontal ligament (PDL)-bone and PDL-cementum attachment sites and within the tissues per se. DESIGN: Accentuated mechanical strain was induced by applying a unidirectional force of 0.06N for 14 days on molars in a rat model. The associated changes in functional space between tooth and bone, mineral formation and resorbing events at the PDL-bone and PDL-cementum attachment sites were identified by using micro-X-ray computed tomography (micro-XCT), atomic force microscopy (AFM), dynamic histomorphometry, Raman microspectroscopy, AFM-based nanoindentation technique, and were correlated with histochemical stains specific to low and high molecular weight GAGs, including biglycan, and osteoclast distribution through tartrate-resistant acid phosphatase (TRAP) staining. RESULTS: Unique chemical and mechanical qualities including heterogenous bony fingers with hygroscopic Sharpey's fibers contributing to a higher organic (amide III - 1240 cm-1) to inorganic (phosphate - 960 cm-1) ratio, with lower average elastic modulus of 8 GPa versus 12 GPa in unadapted regions were identified. Furthermore, an increased presence of elemental Zn in cement lines and mineralizing fronts of PDL-bone was observed. Adapted regions containing bony fingers exhibited woven bone-like architecture and these regions rich in biglycan (BGN) and bone sialoprotein (BSP) also contained high-molecular weight polysaccharides predominantly at the site of polarized bone growth. CONCLUSIONS: From a fundamental science perspective the shift in local properties due to strain amplification at the soft-hard tissue attachment sites is governed by semiautonomous cellular events at the PDL-bone and PDL-cementum sites. Over time, these strain-mediated events can alter the physicochemical properties of tissues per se, and consequently the overall biomechanics of the bone-PDL-tooth complex. From a clinical perspective, the shifts in magnitude and duration of forces on the periodontal ligament can prompt a shift in physiologic mineral apposition in cementum and alveolar bone albeit of an adapted quality owing to the rapid mechanical translation of the tooth.

The plastic nature of the human bone-periodontal ligament-tooth fibrous joint.

This study investigates bony protrusions within a narrowed periodontal ligament space (PDL-space) of a human bone-PDL-tooth fibrous joint by mapping structural, biochemical, and mechanical heterogeneity. Higher resolution structural characterization was achieved via complementary atomic force microscopy (AFM), nano-transmission X-ray microscopy (nano-TXM), and microtomography (MicroXCT™). Structural heterogeneity was correlated to biochemical and elemental composition, illustrated via histochemistry and microprobe X-ray fluorescence analysis (µ-XRF), and mechanical heterogeneity evaluated by AFM-based nanoindentation. Results demonstrated that the narrowed PDL-space was due to invasion of bundle bone (BB) into PDL-space. Protruded BB had a wider range with higher elastic modulus values (2-8GPa) compared to lamellar bone (0.8-6GPa), and increased quantities of Ca, P and Zn as revealed by µ-XRF. Interestingly, the hygroscopic 10-30µm interface between protruded BB and lamellar bone exhibited higher X-ray attenuation similar to cement lines and lamellae within bone. Localization of the small leucine rich proteoglycan biglycan (BGN) responsible for mineralization was observed at the PDL-bone interface and around the osteocyte lacunae. Based on these results, it can be argued that the LB-BB interface was the original site of PDL attachment, and that the genesis of protruded BB identified as protrusions occurred as a result of shift in strain. We emphasize the importance of bony protrusions within the context of organ function and that additional study is warranted.

Imaging an adapted dentoalveolar complex.

Adaptation of a rat dentoalveolar complex was illustrated using various imaging modalities. Micro-X-ray computed tomography for 3D modeling, combined with complementary techniques, including image processing, scanning electron microscopy, fluorochrome labeling, conventional histology (H&E, TRAP), and immunohistochemistry (RANKL, OPN) elucidated the dynamic nature of bone, the periodontal ligament-space, and cementum in the rat periodontium. Tomography and electron microscopy illustrated structural adaptation of calcified tissues at a higher resolution. Ongoing biomineralization was analyzed using fluorochrome labeling, and by evaluating attenuation profiles using virtual sections from 3D tomographies. Osteoclastic distribution as a function of anatomical location was illustrated by combining histology, immunohistochemistry, and tomography. While tomography and SEM provided past resorption-related events, future adaptive changes were deduced by identifying matrix biomolecules using immunohistochemistry. Thus, a dynamic picture of the dentoalveolar complex in rats was illustrated.

Matrix metalloproteinase-13 is required for osteocytic perilacunar remodeling and maintains bone fracture resistance.

Like bone mass, bone quality is specified in development, actively maintained postnatally, and disrupted by disease. The roles of osteoblasts, osteoclasts, and osteocytes in the regulation of bone mass are increasingly well defined. However, the cellular and molecular mechanisms by which bone quality is regulated remain unclear. Proteins that remodel bone extracellular matrix, such as the collagen-degrading matrix metalloproteinase (MMP)-13, are likely candidates to regulate bone quality. Using MMP-13-deficient mice, we examined the role of MMP-13 in the remodeling and maintenance of bone matrix and subsequent fracture resistance. Throughout the diaphysis of MMP-13-deficient tibiae, we observed elevated nonenzymatic cross-linking and concentric regions of hypermineralization, collagen disorganization, and canalicular malformation. These defects localize to the same mid-cortical bone regions where osteocyte lacunae and canaliculi exhibit MMP-13 and tartrate-resistant acid phosphatase (TRAP) expression, as well as the osteocyte marker sclerostin. Despite otherwise normal measures of osteoclast and osteoblast function, dynamic histomorphometry revealed that remodeling of osteocyte lacunae is impaired in MMP-13(-/-) bone. Analysis of MMP-13(-/-) mice and their wild-type littermates in normal and lactating conditions showed that MMP-13 is not only required for lactation-induced osteocyte perilacunar remodeling, but also for the maintenance of bone quality. The loss of MMP-13, and the resulting defects in perilacunar remodeling and matrix organization, compromise MMP-13(-/-) bone fracture toughness and postyield behavior. Taken together, these findings demonstrate that osteocyte perilacunar remodeling of mid-cortical bone matrix requires MMP-13 and is essential for the maintenance of bone quality.