Johanna Bolander

Current views on calcium phosphate osteogenicity and the translation into effective bone regeneration strategies

Calcium phosphate (CaP) has traditionally been used for the repair of bone defects because of its strong resemblance to the inorganic phase of bone matrix. Nowadays, a variety of natural or synthetic CaP-based biomaterials are produced and have been extensively used for dental and orthopaedic applications. This is justified by their biocompatibility, osteoconductivity and osteoinductivity (i.e. the intrinsic material property that initiates de novo bone formation), which are attributed to the chemical composition, surface topography, macro/microporosity and the dissolution kinetics. However, the exact molecular mechanism of action is unknown. This review paper first summarizes the most important aspects of bone biology in relation to CaP and the mechanisms of bone matrix mineralization. This is followed by the research findings on the effects of calcium (Ca²⁺) and phosphate (PO₄³⁻) ions on the migration, proliferation and differentiation of osteoblasts during in vivo bone formation and in vitro culture conditions. Further, the rationale of using CaP for bone regeneration is explained, focusing thereby specifically on the materials osteoinductive properties. Examples of different material forms and production techniques are given, with the emphasis on the state-of-the art in fine-tuning the physicochemical properties of CaP-based biomaterials for improved bone induction and the use of CaP as a delivery system for bone morphogenetic proteins. The use of computational models to simulate the CaP-driven osteogenesis is introduced as part of a bone tissue engineering strategy in order to facilitate the understanding of cell-material interactions and to gain further insight into the design and optimization of CaP-based bone reparative units. Finally, limitations and possible solutions related to current experimental and computational techniques are discussed.

Healing of a Large Long-Bone Defect through Serum-Free In Vitro Priming of Human Periosteum-Derived Cells

Summary Clinical translation of cell-based strategies for regenerative medicine demands predictable in vivo performance where the use of sera during in vitro preparation inherently limits the efficacy and reproducibility. Here, we present a bioinspired approach by serum-free pre-conditioning of human periosteum-derived cells, followed by their assembly into microaggregates simultaneously primed with bone morphogenetic protein 2 (BMP-2). Pre-conditioning resulted in a more potent progenitor cell population, while aggregation induced osteochondrogenic differentiation, further enhanced by BMP-2 stimulation. Ectopic implantation displayed a cascade of events that closely resembled the natural endochondral process resulting in bone ossicle formation. Assessment in a critical size long-bone defect in immunodeficient mice demonstrated successful bridging of the defect within 4 weeks, with active contribution of the implanted cells. In short, the presented serum-free process represents a biomimetic strategy, resulting in a cartilage tissue intermediate that, upon implantation, robustly leads to the healing of a large long-bone defect.

Bioinspired seeding of biomaterials using three dimensional microtissues induces chondrogenic stem cell differentiation and cartilage formation under growth factor free conditions

Cell laden biomaterials are archetypically seeded with individual cells and steered into the desired behavior using exogenous stimuli to control growth and differentiation. In contrast, direct cell-cell contact is instructive and even essential for natural tissue formation. Namely, microaggregation and condensation of mesenchymal progenitor cells triggers chondrogenesis and thereby drives limb formation. Yet a biomimetic strategy translating this approach into a cell laden biomaterial-based therapy has remained largely unexplored. Here, we integrate the microenvironment of cellular condensation into biomaterials by encapsulating microaggregates of a hundred human periosteum-derived stem cells. This resulted in decreased stemness-related markers, up regulation of chondrogenic genes and improved in vivo cartilage tissue formation, as compared to single cell seeded biomaterials. Importantly, even in the absence of exogenous growth factors, the microaggregate laden hydrogels outperformed conventional single cell laden hydrogels containing supraphysiological levels of the chondrogenic growth factor TGFB. Overall, the bioinspired seeding strategy described herein represents an efficient and growth factor-free approach to efficiently steer cell fate and drive tissue formation for biomaterial-based tissue engineering strategies.

A Qualitative Model of the Differentiation Network in Chondrocyte Maturation: A Holistic View of Chondrocyte Hypertrophy

Differentiation of chondrocytes towards hypertrophy is a natural process whose control is essential in endochondral bone formation. It is additionally thought to play a role in several pathophysiological processes, with osteoarthritis being a prominent example. We perform a dynamic analysis of a qualitative mathematical model of the regulatory network that directs this phenotypic switch to investigate the influence of the individual factors holistically. To estimate the stability of a SOX9 positive state (associated with resting/proliferation chondrocytes) versus a RUNX2 positive one (associated with hypertrophy) we employ two measures. The robustness of the state in canalisation (size of the attractor basin) is assessed by a Monte Carlo analysis and the sensitivity to perturbations is assessed by a perturbational analysis of the attractor. Through qualitative predictions, these measures allow for an in silico screening of the effect of the modelled factors on chondrocyte maintenance and hypertrophy. We show how discrepancies between experimental data and the model’s results can be resolved by evaluating the dynamic plausibility of alternative network topologies. The findings are further supported by a literature study of proposed therapeutic targets in the case of osteoarthritis.

The Effect of Activating Fibroblast Growth Factor Receptor 3 Mutations on Osteogenic Differentiation and Ectopic Bone Formation by Human Periosteal Derived Cells

Activating mutations in Fibroblast Growth Factor Receptor 3 (FGFR3) have previously been shown to cause skeletal dysplasias through their effect on growth plate chondrocytes. However, the effect of FGFR3 mutations on bone progenitor cells may differ. The objective of this study was to investigate the effect of specific activating FGFR3 mutations on ectopic in vivo bone formation by periosteal derived cells (PDCs) seeded on calcium phosphate/ collagen scaffolds. PDCs were isolated from hypochondroplasic (N540K mutation) and achondroplasic (G380R mutation) patients, along with age/sex matched controls. These cells were characterised in vitro for proliferation, osteogenic differentiation, FGFR3 signalling and in vivo bone formation. Subsequently, empirical modelling was used to find correlations between in vivo formed bone and in vitro cell behaviour. These data showed that in contrast to the G380R mutation, which produced no bone, the N540K mutation induced significant ectopic bone formation on specific carriers. This allowed correlation between percentage of induced bone formation to elevated in vitro proliferation and differentiation. Correlating osteogenic markers included Collagen type 1, alkaline phosphatase and osteocalcin. Enhanced proliferation was attributed to increased phosphorylation of Erk-1/2.

Toward Advanced Therapy Medicinal Products (ATMPs) Combining Bone Morphogenetic Proteins (BMP) and Cells for Bone Regeneration

Cell-based implants with or without osteoinductive biomolecules on optimal carrier materials as an advanced therapeutic medicinal product (ATMP) are a promising strategy for poorly healing long-bone defects. This chapter will focus on ATMPs combining bone morphogenetic proteins (BMPs) and progenitor cells for the clinical treatment of large bone defects in compromised environments. We describe BMP signaling involved in the process of bone fracture healing with specific emphasis on clinically relevant BMP ligands, followed by characterization and BMP responsiveness of progenitor cells obtained from different sources. Then we explore different biomaterials and their contribution to achieve optimal BMP release and osteoinduction. Finally, we provide a perspective on the applicability of ATMPs in bone repair by reviewing the preclinical studies carried out so far in various animal models. We believe the era of regenerative medicine has just started. First-generation BMP and stem cell technologies have demonstrated that in the postnatal environment, one can successfully enhance the healing of damaged tissues by recapitulating the principles of developmental tissue formation. A second generation of products is needed that leads to successful bone healing in compromised environments.