Matilde Bongio

Development of bone substitute materials: from ‘biocompatible’ to ‘instructive’

Progress made in basic research in the last decades led to a tremendous increase in quality of clinically applied bone substitute materials (polymers, ceramics and composites). The desired biological performance of these materials has consequently shifted from a passive role where materials were merely accepted by the body to an active role in which materials instruct their biological surroundings. Bone substitute materials were traditionally based on bioceramics, that can be optimized in terms of composition, structure and porosity. Now, polymers are increasingly gaining importance for use in medical applications due to their high versatility. This review provides an overview of the evolution from 1st generation biotolerant and bioinert materials via 2nd generation bioresponsive bone substitutes towards 3rd generation bioinstructive bone substitute materials that possess inherent biological cues for bone regeneration.

Preclinical evaluation of injectable bone substitute materials

Injectable bone substitutes (IBSs) represent an attractive class of ready‐to‐use biomaterials, both ceramic‐ and polymer‐based, as they offer the potential benefit of minimally invasive surgery and optimal defect filling. Although in vitro assessments are the first step in the process of development, the safety and efficacy of an IBS strongly depend on validated preclinical research prior to clinical trials. However, the selection of a suitable preclinical model for performance evaluation of an IBS remains a challenge, as no gold standard exists to define the best animal model. In order to succeed in this attempt, we identified three stages of development, including (a) proof‐of‐principle, (b) predictive validity and (c) general scientific legitimacy, and the respective criteria that should be applied for such selection. The second part of this review provides an overview of commonly used animals for IBSs. Specifically, scientific papers published between January 1996 and March 2012 were retrieved that report the use of preclinical models for the evaluation of IBSs in situations requiring bone healing and bone augmentation. This review is meant not only to describe the currently available preclinical models for IBS application, but also to address critical considerations of such multi‐factorial evaluation models (including animal species, strain, age, anatomical site, defect size and type of bone), which can be indicative but in most cases edge away from the human reality. Consequently, the ultimate goal is to guide researchers toward a more careful and meaningful interpretation of the results of experiments using animal models and their clinical applications. Copyright

Subcutaneous tissue response and osteogenic performance of calcium phosphate nanoparticle-enriched hydrogels in the tibial medullary cavity of guinea pigs

In the current study, oligo(poly(ethylene glycol) fumarate) (OPF)-based hydrogels were tested for the first time as injectable bone substitute materials. The primary feature of the material design was the incorporation of calcium phosphate (CaP) nanoparticles within the polymeric matrix in order to compare the soft tissue response and bone-forming capacity of plain OPF hydrogels with CaP-enriched OPF hydrogel composites. To that end, pre-set scaffolds were implanted subcutaneously, whereas flowable polymeric precursor solutions were injected in a tibial ablation model in guinea pigs. After 8 weeks of implantation, histological and histomorphometrical evaluation of the subcutaneous scaffolds confirmed the biocompatibility of both types of hydrogels. Nevertheless, OPF hydrogels presented a loose structure, massive cellular infiltration and extensive material degradation compared to OPF-CaP hydrogels that were more compact. Microcomputed tomography and histological and histomorphometrical analyses showed comparable amounts of new trabecular bone in all tibias and some material remnants in the medial and distal regions. Particularly, highly calcified areas were observed in the distal region of OPF-CaP-treated tibias, which indicate a heterogeneous distribution of the mineral phase throughout the hydrogel matrix. This phenomenon can be attributed to either hindered gelation under highly perfused in vivo conditions or a faster degradation rate of the polymeric hydrogel matrix compared to the nanostructured mineral phase, resulting in loss of entrapment of the CaP nanoparticles and subsequent sedimentation.

Processing and in vivo evaluation of multiphasic calcium phosphate cements with dual tricalcium phosphate phases.

Calcium phosphate cements (CPCs) use the simultaneous presence of several calcium phosphates phases. This is done to generate specific bulk and in vivo properties. This work has processed and evaluated novel multiphasic CPCs containing dual tricalcium phosphate (TCPs) phases. Dual TCPs containing α- and β-TCP phases were obtained by thermal treatment. Standard CPC (S-CPC) was composed of α-TCP, anhydrous dicalcium phosphate and precipitated hydroxyapatite, while modified CPC (DT-CPC) included both α- and β-TCP. Physicochemical characterization of these CPCs was based on scanning electron microscopy, X-ray diffraction, specific surface area (SSA) and particle size (PS) analysis and mechanical properties. This characterization allowed the selection of one DT-CPC for setting time, cohesion and biological assessment compared with S-CPC. Biological assessment was carried out using a tibial intramedullary cavity model and subcutaneous pouches in guinea pigs. Differences in the surface morphology and crystalline phases of the treated TCPs were detected, although PS analysis of the milled CPC powders produced similar results. SSA analysis was significantly higher for DT-CPC with α-TCP treated at 1100°C for 5h. Poorer mechanical properties were found for DT-CPC with α-TCP treated at 1000°C. Setting time and cohesion, as well as the in vivo performance, were similar in the selected DT-CPC and the S-CPC. Both CPCs created the desired host reactions in vivo.

Effect of Nano-HA/Collagen Composite Hydrogels on Osteogenic Behavior of Mesenchymal Stromal Cells

This study aimed to comparatively evaluate the in vitro effect of nanosized hydroxyapatite and collagen (nHA/COL) based composite hydrogels (with different ratios of nHA and COL) on the behavior of human mesenchymal stromal cells (MSCs), isolated from either adipose tissue (AT-MSCs) or bone marrow (BM-MSCs). We hypothesized that (i) nHA/COL composite hydrogels would promote the osteogenic differentiation of MSCs in an nHA concentration dependent manner, and that (ii) AT-MSCs would show higher osteogenic potential compared to BM-MSCs, due to their earlier observed higher proliferation and osteogenic differentiation potential in 2D in vitro cultures [1]. The obtained results indicated that AT-MSCs show indeed high proliferation, differentiation and mineralization capacities in nHA/COL constructs compared to BM-MSCs, but this effect was irrespective of nHA concentration. Based on the results of alkaline phosphatase (ALP) activity and osteocalcin (OCN) protein level, the osteogenic differentiation of BM-MSCs started in the beginning of the culture period and for AT-MSCs at the end of the culture period. At a molecular level, both cell types showed high expression of osteogenic markers (bone morphogenic protein 2 [BMP2], runt-related transcription factor 2 [RUNX2], OCN or COL1) in both an nHA concentration and time dependent manner. In conclusion, AT-MSCs demonstrated higher osteogenic potential in nHA/COL based 3D micro-environments compared to BM-MSCs, in which proliferation and osteogenic differentiation were highly promoted in a time dependent manner, irrespective of nHA amount in the constructs. The fact that AT-MSCs showed high proliferation and mineralization potential is appealing for their application in future pre-clinical research as an alternative cell source for BM-MSCs.

In vitro and in vivo enzyme-mediated biomineralization of oligo(poly(ethylene glycol) fumarate hydrogels.

The enzyme alkaline phosphatase (ALP) is added at different concentrations (i.e., 0, 2.5, and 10 mg   ml(-1) ) to oligo(poly(ethylene glycol)fumarate) (OPF) hydrogels. The scaffolds are either incubated in 10 mM calcium glycerophosphate (Ca-GP) solution for 2 weeks or implanted in a rat subcutaneous model for 4 weeks. Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), and alizarin red staining show a strong ability to form minerals exclusively in ALP-containing hydrogels in vitro. Additionally, the calcium content increases with increasing ALP concentration. Similarly, only ALP-containing hydrogels induce mineralization in vivo. Specifically, small (≈5-20 µm) mineral deposits are observed at the periphery of the hydrogels near the dermis/scaffold interface using Von Kossa and alizarin red staining.

Comparison of cell-loading methods in hydrogel systems

Bone regenerative medicine, based on the combined use of cells and scaffolds, represents a promising strategy in bone regeneration. Hydrogels have attracted huge interests for application as a scaffold for minimally invasive surgery. Collagen and oligo(poly(ethylene glycol)fumarate) (OPF) hydrogels are the representatives of two main categories of hydrogels, that is, natural- and synthetic-based hydrogels. With these the optimal cell-loading (i.e., cell distribution inside the hydrogels) method was assessed. The cell behavior of both bone marrow- and adipose tissue-derived mesenchymal stem cells (BM- and AT-MSCs) in three loading methods, which are dispersed (i.e., homogeneous cell encapsulation, D), sandwich (i.e., cells located in between two hydrogel layers, S), and spheroid (i.e., cell pellets encapsulation, Sp) loading in two hydrogel systems (i.e., collagen and OPF), was compared. The results suggested that the cell behavior was influenced by the hydrogel type, meaning cells cultured in collagen hydrogels had higher proliferation and osteogenic differentiation capacity than in OPF hydrogels. In addition, AT-MSCs exhibited higher proliferation and osteogenic properties compared to BM-MSCs. However, no difference was observed for mineralization among the three loading methods, which did not approve the hypothesis that S and Sp loading would increase osteogenic capacity compared to D loading. In conclusion, D and Sp loading represents two promising cell loading methods for injectable bone substitute materials that allow application of minimally invasive surgery for cell-based regenerative treatment.