Yu Ling Zhang

In vitro degradation and in vivo resorption of dicalcium phosphate cement based grafts

There are two types of DCP: dihydrated (brushite) and anhydrous (monetite). After implantation, brushite converts to hydroxyapatite (HA) which resorbs very slowly. This conversion is not observed after implantation of monetite cements and result in a greater of resorption. The precise mechanisms of resorption and degradation however of these ceramics remain uncertain. This study was designed to investigate the effect of: porosity, surface area and hydration on in vitro degradation and in vivo resorption of DCP. Brushite and two types of monetite cement based grafts (produced by wet and dry thermal conversion) were aged in phosphate buffered saline (PBS) and bovine serum solutions in vitro and were implanted subcutaneously in rats. Here we show that for high relative porosity grafts (50-65%), solubility and surface area does not play a significant role towards in vitro mass loss with disintegration and fragmentation being the main factors dictating mass loss. For grafts having lower relative porosity (35-45%), solubility plays a more crucial role in mass loss during in vitro ageing and in vivo resorption. Also, serum inhibited dissolution and the formation of HA in brushite cements. However, when aged in PBS, brushite undergoes phase conversion to a mixture of octacalcium phosphate (OCP) and HA. This phase conversion was not observed for monetite upon ageing (in both serum and PBS) or in subcutaneous implantation. This study provides greater understanding of the degradation and resorption process of DCP based grafts, allowing us to prepare bone replacement materials with more predictable resorption profiles.

Hydrocaffeic acid–chitosan nanoparticles with enhanced stability, mucoadhesion and permeation properties

Catechol-containing molecules, such as hydrocaffeic acid (HCA) have been shown to increase the mucoadhesion of several polymers. We report here a simple and bioinspired approach to enhance chitosan (CS) mucoadhesion and stabilize it in nanoparticulate form by preparing HCA-CS conjugates. HCA-CS conjugates containing 6 and 15mol% HCA were synthesized and characterized by FT-IR, (1)H NMR and UV-vis spectrophotometry. HCA-CS nanoparticles prepared by ionic gelation with sodium tripolyphosphate (TPP) ranged in size between 100 and 250nm depending on the polymer and TPP/CS weight ratio. In contrast to CS nanoparticles, which aggregate at pH>6.5, HCA-CS nanoparticles did not show any sign of aggregation or precipitation over the 4-10 pH range and maintain their size. Unexpectedly, HCA-CS nanoparticles also maintained their size and polydispersity index at pH 7.4 and NaCl concentrations of up to 500mM. Partial oxidation of HCA resulted in nanoparticle cross-linking and improved stability at pH<4. HCA-CS mucoadhesion to rabbit small intestine was 6 times higher than unmodified CS. CS and HCA-CS nanoparticles were able to induce reversible tight junction opening in Caco-2 cell monolayers. Tight junction opening facilitated the permeability of a model hydrophilic molecule, fluorescein isothiocyanate-labeled dextran (FD4) and was 3 times higher in the cells treated with HCA-CS 15% nanoparticles compared to control groups. HCA-CS conjugates were found to be excellent candidates for stable nanodelivery systems with enhanced oral absorption of hydrophilic molecules.

Two-Dimensional Magnesium Phosphate Nanosheets Form Highly Thixotropic Gels That Up-Regulate Bone Formation

Hydrogels composed of two-dimensional (2D) nanomaterials have become an important alternative to replace traditional inorganic scaffolds for tissue engineering. Here, we describe a novel nanocrystalline material with 2D morphology that was synthesized by tuning the crystallization of the sodium-magnesium-phosphate system. We discovered that the sodium ion can regulate the precipitation of magnesium phosphate by interacting with the crystals surface causing a preferential crystal growth that results in 2D morphology. The 2D nanomaterial gave rise to a physical hydrogel that presented extreme thixotropy, injectability, biocompatibility, bioresorption, and long-term stability. The nanocrystalline material was characterized in vitro and in vivo and we discovered that it presented unique biological properties. Magnesium phosphate nanosheets accelerated bone healing and osseointegration by enhancing collagen formation, osteoblasts differentiation, and osteoclasts proliferation through up-regulation of COL1A1, RunX2, ALP, OCN, and OPN. In summary, the 2D magnesium phosphate nanosheets could bring a paradigm shift in the field of minimally invasive orthopedic and craniofacial interventions because it is the only material available that can be injected through high gauge needles into bone defects in order to accelerate bone healing and osseointegration.

Fibril formation pH controls intrafibrillar collagen biomineralization in vitro and in vivo

We demonstrate that intrafibrillar, homogenous collagen biomineralization can be achieved by controlling self-assembly under mildly alkaline conditions. Using dense collagen (DC) gels as an osteoid model, we modulated their fibrillogenesis environment to evaluate the effects of fibrillogenesis pH on the protein charge distribution and ultimately on biomineralization. Cationic and anionic dye staining and electron cryomicroscopy analyses established that fibrillogenesis under mildly alkaline conditions promotes the formation of electronegative charges within the protein (anionic DC gels). These charges are stable upon titration of the gel pH to physiological values. Subsequent exposure of anionic DC gels to simulated body fluid induced the intrafibrillar biomineralization of the gels, promoting a rapid, extensive formation of carbonated hydroxyapatite, and strongly impacting gel mechanical properties. The generality and significance of this approach has been addressed by implanting freshly made anionic DC gels in vivo, in a rat subcutaneous model. Subcutaneous implants showed an extensive, homogenous biomineralization as early as at day 7, indicating that anionic collagen gels rapidly self-mineralize upon contact with body fluids in a non-osseous implantation site. The control of collagen fibrillogenesis pH provides not only new interpretations to what has been called the collagen mineralization enigma by demonstrating that neat collagen can intrafibrilarly self-mineralize, but it will also set a new starting point for the use of DC gels in bone regenerative medicine, in addition as potential applications as mineralized tissue model or as slow-release delivery carriers.

Controlling Bone Graft Substitute Microstructure to Improve Bone Augmentation

Vertical bone augmentation procedures are frequently carried out to allow successful placement of dental implants in otherwise atrophic ridges and represent one of the most common bone grafting procedures currently performed. Onlay autografting is one of the most prevalent and predictable techniques to achieve this; however, there are several well documented complications and drawbacks associated with it and synthetic alternatives are being sought. Monetite is a bioresorbable dicalcium phosphate with osteoconductive and osteoinductive potential that has been previously investigated for onlay bone grafting and it is routinely made by autoclaving brushite to simultaneously sterilize and phase convert. In this study, monetite disc-shaped grafts are produced by both wet and dry heating methods which alter their physical properties such as porosity, surface area, and mechanical strength. Histological observations after 12 weeks of onlay grafting on rabbit calvaria reveal higher bone volume (38%) in autoclaved monetite grafts in comparison with the dry heated monetite grafts (26%). The vertical bone height gained is similar for both the types of monetite grafts (up to 3.2 mm). However, it is observed that the augmented bone height is greater in the lateral than the medial areas of both types of monetite grafts. It is also noted that the higher porosity of autoclaved monetite grafts increases the bioresorbability, whereas the dry heated monetite grafts having lower porosity but higher surface area resorb to a significantly lesser extent. This study provides information regarding two types of monetite onlay grafts prepared with different physical properties that can be further investigated for clinical vertical bone augmentation applications.

A new class of bioactive glasses: Calcium–magnesium sulfophosphates

Low-melting ionic sulfophosphate glasses from the system P2O5-SO4-MO-Na2O (M = Zn(2+), Ca(2+) or Mg(2+)) have been previously shown by us to allow tuneable aqueous dissolution and also enable processing temperatures well below 400°C. Sulfate ions are extremely safe for use in the body as decades of use of calcium sulfate bone grafts testifies and there is no known limit on their adult oral toxicity. This glass system therefore offers great potential for use as biomaterials, especially in organic-inorganic hybrid systems such as glass-polymer composites for tissue engineering or drug encapsulation and delivery applications. A compositional region was identified where stable sulfophosphates of the type P2O5-SO4-(Ca, Mg, Zn)O-Na2O can be fabricated. For these glasses, the viscosity-temperature-dependence, glass transformation temperatures (Tg ) and the onset of crystallization were evaluated as the primary processing parameters. As a first step in exploring their potential as a biomaterial, in this study we examine the bioactivity of several compositions of these glasses using fibroblast, monocyte, and osteoclast cell culture models to determine cellular responses in terms of attachment, proliferation, differentiation, and toxicity.

Effect of processing conditions of dicalcium phosphate cements on graft resorption and bone formation

Dicalcium phosphate cements (brushite and monetite) are resorbable biomaterials with osteoconductive potential for bone repair and regeneration that have yet to gain widespread commercial use. Brushite can be converted to monetite by heat treatments additionally resulting in various changes in the physico-chemical properties. However, since conversion is most commonly performed using autoclave sterilisation (wet heating), it is uncertain whether the properties observed for monetite as a result of heating brushite under dry conditions affect resorption and bone formation favourably. This study was designed to produce monetite grafts of differing physical form by autoclaving and dry heating (under vacuum) to be compared with brushite biomaterials in an orthotopic pre-clinical implantation model in rabbit for 12weeks. It was observed that monetite grafts had higher porosity and specific surface area than their brushite precursors. The autoclaved monetite grafts had compressive strength reduced by 50% when compared with their brushite precursors. However, the dry heat converted monetite grafts had compressive strength comparable with brushite. Results from in vivo experiments revealed that both types of monetite graft materials resorbed faster than brushite and more bone formation was achieved. There was no significant difference in the amount of bone formed between the two types of monetite grafts. The implanted brushite grafts underwent phase transformation to form hydroxyapatite, which ultimately limited bioresorption. However, this was not observed in both types of monetite grafts. In summary, both autoclaving and dry heating the preset brushite cement grafts resulted in monetite biomaterials which were more resorbable with potential to be investigated and optimized for orthopaedic and maxillofacial bone repair and regeneration applications. STATEMENT OF SIGNIFICANCE We present in this original research article a comparison between dicalcium phosphate cement based grafts (brushite and 2 types of monetite grafts prepared by wet and dry thermal processing) with regards to resorption and bone formation in vivo after orthotopic implantation in rabbit condylar femural region. To the best of our knowledge this is the first in vivo study that reports a comparison resorption and bone formation using brushite and two types of monetite biomaterials. Also, we have included in the manuscript a summary of all the in vivo studies performed on brushite and monetite biomaterials to date. This includes cement composition, physical properties (porosity and surface area), implantation and histomorphometrical details such as animal species, site of implantation, observation period, percentage bone tissue formation and residual graft material. In addition, we calculated the percentage resorption of graft materials based upon various implantation sites and included that into the discussion section. The results of this original research provides greater understanding of the resorption processes of dicalcium phosphate based grafts, allowing preparation of bone substitute materials with more predictable resorption profiles in future.

Local delivery of iron chelators reduces in vivo remodeling of a calcium phosphate bone graft substitute

UNLABELLED Iron chelators are known activators of the Hypoxia Includible Factor-1α (HIF-1α) pathway, a critical cellular pathway involved in angiogenic responses to hypoxia. Local delivery of these chelators has shown promise in bone tissue engineering strategies by inducing angiogenesis and osteogenesis. Hypoxic microenvironments are also a stimulus for osteoclast differentiation and resorptive activity, a process likely mediated by HIF-1α. In vitro, low doses of the iron chelator Deferoxamine (DFO) has shown to induce HIF-1α mediated osteoclast formation and function. However other studies have proposed an opposite in vitro effect likely through HIF independent mechanisms. To investigate use of these medications in bioceramic based bone tissue engineering strategies this study aimed to determine the in vivo effect of local delivery of iron chelators on bioceramic remodeling. A non-weight bearing cranial onlay model was used to assess monetite resorption and new bone formation in the presence or absence of a repeated delivery of two iron chelators, DFO and 1,10 Phenanthroline (PHT) at doses known to induce HIF. We found a marked reduction graft resorption and remodeling associated with iron chelation. This was correlated to a 3-fold reduction in osteoclast number at the bone graft interface. Iron is needed for mitochondrial biogenesis during osteoclastic differentiation and reducing extracellular iron levels may inhibit this process and possibly overpower any HIF induced osteoclast formation. Our findings suggest that these inexpensive and widely available molecules may be used to locally reduce bioceramic scaffold resorption and encourages future investigations of iron chelators as bone anti-resorptive agents in other clinical contexts. STATEMENT OF SIGNIFICANCE Low doses of iron chelators can induce angiogenesis and osteogenesis in repairing bone by stimulating the oxygen sensitive gene; hypoxia inducible factor. These medications have potential to augment bioceramic based bone tissue engineering strategies without the downsides of protein-based growth factors. HIF activation is also known to stimulate osteoclast-mediated resorption and could potentially accelerate remodeling of biocermaics, however we have shown that the local delivery of iron chelation at doses known to induce HIF resulted in a reduction of monetite resorption and a significant decrease in osteoclast number at the bone graft interface. This maybe due to HIF independent mechanism. This is the first study to show a local effect of iron chelators in vivo on osteoclast-mediated resorption. This opens the potential of further study of these bifunctional medications to modulate resorption of biocermaics in environments where a prolonged presence of material is desired for graft site stability. Moreover these safe widely used medications can be explored to locally reduce osteoclasts in pathological bone resorption.

Intra-tumor delivery of zoledronate mitigates metastasis-induced osteolysis superior to systemic administration.

Bisphosphonates (BPs) have recently been shown to have direct anti-tumor properties. Systemic treatment with BPs can have multiple adverse effects such as osteonecrosis of the jaw and BP induced bone fracturing and spine instability. While benefits of systemic BP treatments may outweigh risks, local treatment with BPs has been explored as an alternate strategy to reduce unwarranted risk. In the present study, we examined whether local delivery of BPs inhibits tumor-induced osteolysis and tumor growth more effectively than systemic treatment in an animal model of tumor-induced bone disease. Following establishment of an intra-tibial model of bone metastases in athymic mice, the experimental group was treated by local administration of zoledronate into the tibial lesion. A comparison of the effect of local versus systemic delivery of zoledronate on the formation of tumor-induced osteolysis was also carried out. A significant increase in mean bone volume/tissue volume % (BV/TV) of the locally treated group (12.30±2.80%) compared to the control group (7.13±1.22%) (P<0.001). Additionally, there was a significant increase in the BV/TV (10.90±1.25%) in the locally treated group compared to the systemically treated group (7.53±0.75%) (P=0.005). These preliminary results suggest that local delivery of BPs outperforms both systemic and control treatments to inhibit tumor-induced osteolysis.