Mineralized collagen scaffolds for regenerative engineering applications

Abstract

Collagen is a primary constituent of the tissue extracellular matrix. As a result, collagen has been a common component of tissue engineering biomaterials, including those to promote bone regeneration or to investigate cell-material interactions in the context of bone homeostasis or disease. This review summarizes key considerations regarding current state-of-the-art design and use of collagen biomaterials for these applications. We also describe strategic opportunities for collagen biomaterials to address a new era of challenges, including immunomodulation and appropriate consideration of sex and other patient characteristics in biomaterial design.

Introduction

Critical-sized extremity and craniomaxillofacial bone defects pose an unmet clinical challenge as they cannot heal naturally and instead require surgical intervention [1,2]. Clinical approaches for defect repair include autografts and allografts, which can be inadequate due to limited bone availability and secondary site morbidity [3]. Graft alternatives, such as metals, bioglass ceramics, and polymers, display limited bioactivity and biodegradation with acidic by-products that activate inflammatory reactions that often lead to implant rejection in vivo [4,5].

Collagen biomaterials provide an opportunity to overcome these challenges. A key element of the native bone extracellular matrix, its use offers the ability to promote pro-regenerative cell activity. Our lab had developed a class of mineralized collagen (MC) scaffolds that enhance bone regeneration without the need for exogenously added stem cells and growth factors (e.g. BMP-2). The tunable structure, composition, and mechanics of these porous MC biomaterials makes it possible to address a series of unanswered questions in the context of bone repair. In this review, we will discuss how MC scaffolds have been implemented in vitro and in vivo to identify surgically accessible approaches for critical-size bone repair. We will further discuss design principles for next-generation implants as well as their use as advanced model systems to accelerate the translation of these biomaterials to the clinic.

Heterogeneity in the bone microenvironment

Bone is a multiscale tissue with a hierarchical structure that can inspire biomaterial design. Bone consists of 60% inorganic components (with type-I collagen > 90%), which are primarily hydroxyapatite mineral crystals, 10% water, and 30% organic components. However, the relative amount of these constituents can vary based on age, site in the body, gender, ethnicity, and health status [6–10]. Beyond its complex structure, bone is a dynamic tissue, which, following injury, hosts a spatiotemporally heterogeneous population of cells, including osteoprogenitors, osteoblasts, osteoclasts, immune cells, and vascular cells [11•]. These cells are constantly engaging in multicellular dialog influencing each other’s behavior. Immune cells such as macrophages are some of the first responders to the site of injury and can polarize to a gradient of phenotypes. These cells are responsible for the clearance of debris, recruitment and support of new vasculature, and recruitment of stem cells. Stem cells that arrive at the site of injury can differentiate toward the osteoblastic lineage, while recruited monocytes can differentiate to osteoclasts, the bone-resorbing cell. Osteoblasts and osteoclasts act in concert to remodel bone and achieve a state of equilibrium called homeostasis. All these cell types play critical roles in bone repair and the absence of a single cell type would inhibit the others from functioning normally and regenerate bone.

Advancing biomaterial approaches for bone regeneration

A prevalent strategy in biomaterials’ design for bone regeneration has been enhancing mesenchymal stem cell (MSC) osteogenic capacity. Thus, approaches have sought to enhance MSC invasion, proliferation, and osteogenic differentiation through the use of exogenously added growth factors such as BMP-2 [12–14]. However, the need for supraphysiological doses of such exogenously added factors has also driven adverse effects such as inflammation, ectopic bone formation, and increased osteoclast resorption resulting in such approaches being taken off the market [15]. And while nonMC scaffolds have been used successfully in the clinic to promote dermal and peripheral nerve regeneration, there is a significant opportunity to adapt these materials for musculoskeletal applications [16,17]. Hence, recent efforts have shifted to identifying materials that provide signals to elicit endogenous production of growth factors by recruited MSCs. However, these approaches fail to consider the complexity of the bone injury microenvironment, notably reciprocal crosstalk interactions between MSCs, immune cells, and vascular cells that can dynamically alter MSC osteogenic capacity and the resultant regenerative capacity. Thus, it is pivotal that biomaterial design pivots its approach to focus on understanding and subsequently exploiting multicellular interactions.

Mineralized collagen scaffolds provide a platform to enhance bone regeneration

MC scaffolds are a class of biomaterials comprising type-I collagen, glycosaminoglycans, and precipitated brushite mineral. Fabricated via lyophilization from a liquid suspension to form a highly porous microarchitecture, their material architecture, composition, and mechanical properties can be tuned individually [18,19•]. As a result, they are a reproducible platform that has been exploited to examine the role of scaffold design features on MSC osteogenesis and resultant bone regeneration (Figure 1).

Figure 1.

Highlights of MC scaffolds studied for improved bone repair. (a) Anisotropic scaffolds display increased mineral formation compared with isotropic scaffolds [1]. (b) Inclusion of zinc alters mineral microstructure while maintaining osteogenic response [2]. (c) Cross-linked MC scaffolds display greater osteogenic differentiation via elevations in mechanotransduction mediators (YAP/TAZ) compared with non-cross-linked scaffolds [3]. (d) MC scaffolds containing heparin enhance osteogenic response, while anisotropic scaffolds enhance immunomodulatory response [4]. (e) Glycosaminoglycan-mediated MSC secretome differentially influences angiogenic and inflammatory responses [5]. (f) MC scaffolds enhance bone repair without the use of exogenous cells and factors [6]. All figure components were reused with original authors’ permissions.

Scaffold microarchitecture and composition influence mesenchymal stem cell osteogenic response

MC scaffold mechanics (modulus), microarchitecture (pore anisotropy), as well as organic (glycosaminoglycan) and inorganic (mineral architecture) content can instruct MSC osteogenic activity. While MC scaffolds promote MSC osteogenesis in the absence of traditional exogenous supplements, cross-linked and thus stiffer MC scaffolds (3.9+/−0.36 kPa) display greater osteogenic differentiation via elevations in mechanotransduction mediators (YAP/TAZ) and the canonical Wnt signaling pathway compared with non-cross-linked MC scaffolds (0.34+/−0.11 kPa) [20]. Further, anisotropic scaffolds containing aligned tracks of ellipsoidal pores enhance mineral synthesis [18] as well as MSC production of a pro-osteogenic and immunomodulatory hMSC secretome compared with isotropic scaffolds [19•]. Further, scaffolds containing heparin sulfate improve osteogenic responses such as osteoprotegerin (OPG) production and mineral formation, while scaffolds containing chondroitin-6-sulfate enhance hMSC immunomodulatory potential and a sustained osteogenic response [18,19•]. Introduction of zinc sulfate to the MC scaffolds drives formation of a spiky, needle-like, rather than traditional plate-like, brushite mineral without significantly altering pro-osteogenic activity [21]. To improve surgical practicality, it is possible to integrate a 3D-printed polymeric mesh with millimeter-scale porosity into the MC scaffold to form a multiscale composite to increase conformal fitting of complex defects and to differentially promote unique cellular responses [22•].

Immunomodulatory effects of mineralized collagen scaffolds

The inflammatory response can be a decisive factor for implant integration and bone repair, however, only recently has it been considered as a primary design parameter [23•–26]. hMSCs entering the injury microenvironment are exposed to inflammatory stimulation such as pro-inflammatory cytokines and chemokines [27,28]. MC scaffold architecture and composition can bias hMSC responses and function of inflammatory stimuli [19]. Notably, we observed that glycosaminoglycan content, specifically heparin-functionalized MC scaffolds, can enhance hMSC-mediated osteogenesis, while anisotropic scaffolds enhance hMSC immunomodulation and can sustain licensing effects for days after stimulation. Allogeneic tissues, such as placental-derived membranes known to possess antibacterial and antimicrobial properties, can also be incorporated into the MC scaffold. The immunomodulatory potential of these allogeneic tissues does not derive from their soluble products, but rather it is necessary to incorporate membrane-derived matrix into MC scaffolds to promote their osteogenic and immunomodulatory effects [29]. MC scaffolds also provided a 3D environment to demonstrate that hMSC secretome can be biased by scaffold properties and impact monocyte differentiation and endothelial cell tube formation [30]. Notably, chondroitin-6-sulfate-containing MC scaffolds significantly enhanced vascular tube formation and growth, while all MC scaffolds displayed an overall immunosuppressive effect on monocyte differentiation while downregulating osteoclast differentiation.

Clinical translation for mineralized collagen scaffolds

A clinical need for critical-sized bone defects is to enhance early pro-osteogenic activity and the transient reduction of osteoclast resorption to allow for the repair of the defect. MC scaffolds seeded with exogenous hMSCs transduced to increase OPG production promoted osteogenesis while reducing osteoclast resorption [31]. Similar results can be achieved via the direct incorporation of recombinant OPG into the scaffold microarchitecture [32•]. MC scaffolds promoted regenerative healing in critical-sized rabbit cranial defects without the use of exogenously added stem cells or growth factors [33]. Taken together, these data point to the inherent potential of MC scaffolds to be a viable implant strategy for bone regeneration, with the addition of OPG as a viable method to temporally modulate osteoclast activity. Challenges remain regarding large animal model trials, often driven by macroscale mechanical requirement. For example, inclusion of polycaprolactone meshes into MC scaffolds for sub-critical-sized (10-mm dia) porcine mandible defects improved bony ingrowth without the need for exogenous cells, but inclusion of larger volumes of PCL necessary for the mechanical environment of a 25-mm dia. critical-size defect induced more heterogeneity in healing and bone formation [34]. Thus, there remains a critical need to understand key features of load-bearing composites for clinical-scale bone defects.

The next era in bone tissue engineering

There remains a critical need to re-evaluate biomaterials’ design for translation into the clinic (Figure 2) [35,36]. We argue it is essential to shift our basic and clinical research ecosystem to (1) improve our understanding of multicellular dialog and biomaterial interactions at the wound site, and (2) re-evaluate the use of biological variables to more effectively consider patient variability, sex, and underlying health conditions.

Figure 2.

Considerations for the next era of biomaterials for bone regeneration. With greater tunability in material properties, including composition, structure, and mechanics, we need to examine cell response beyond MSCs. Uni- and multicellular response to these materials needs to be examined to understand the dynamic crosstalk interactions that orchestrate repair in the wound microenvironment. However, donor characteristics beyond sex need to be considered as biological variables as experimental observations might not be generalizable across patient populations and thus leaving population groups out of the story. To examine this multitude of variables spanning material, cellular, and patient high-throughput platforms, they need to be developed to rapidly evaluate material–cell interactions in a patient-centric manner. This approach will decrease developmental cost and time and increase clinical translation.

Accelerate the study of multicell dialogs in the wound microenvironment

Platforms that help our community understand the evolution of multicellular crosstalk are essential for more effectively guiding the repair process. The reproducible MC biomaterial environment provides an avenue to evaluate direct cell–cell and paracrine communication pathways, including surface protein–ligand interactions as well as release and trafficking of cytokines, chemokines, and extracellular vesicles (EVs). The potential for MC scaffolds to influence secretion and retention of EVs is particularly an exciting opportunity for growth given to their cargo versatility (e.g. proteins, RNA, and DNA) and their potential as matrix-bound vesicles within a collagenous bone mimicking tissue microenvironment [37–39]. However, such multicellular experiments require two major considerations: 1) choosing the appropriate cell models and 2) minimizing material and cell requirements.

Studies of the immunomodulatory potential of engineered biomaterials often require choosing the appropriate cell model, for example, THP-1 cell line versus primary macrophages. Multiple studies have used THP-1 macrophages due to their affordability and expansion potential, however, THP-1 culture methods vary widely across labs, which will affect comparative consideration of research outputs. Further, THP-1 polarization phenotypes are significantly different than primary macrophages [40,41•]. Primary macrophages, while more clinically relevant, are limited in their expansion and storage capacity, as well as introduce patient-variability concerns.

The shift to primary cells and spatial-omics-based evaluation strategies increasingly calls for novel high-throughput biomaterial approaches. Current high-throughput approaches are often limited to 2D or 2.5D and thus fail to adequately mimic the native bone microenvironment [42,43]. Significant advances need to be made in the fields of tissue engineering and high-throughput platforms to allow for the rapid evaluation of multicell-material interactions.

A call to arms: use patient characteristics as biological variables

It is widely accepted that the defect microenvironment is highly heterogeneous, as a result, our community is making strides to understand this heterogeneity. It is essential we invest similar levels of innovative thinking to understand and address patient heterogeneity [44,45]. A vast amount of literature in the fields of tissue engineering, regenerative medicine, immunology, and stem cell research does not specify patient characteristics as simple as sex or ethnicity (or when they do, the vast majority of patients studied are white males) [46••]. Kurapaty et al. describe the current understanding in the role of sex as a variable in spinal fusion failure illustrating the complexity and limited understanding of sex hormones such as estrogen and androgens pose in bone healing [47]. Furthermore, Gibon et al. discussed the effect of age on the complex cell interactions at the wound site, namely for immune cells and stem cells [48]. However, there is minimal literature discussing the effects of ethnicity, ancestry, and health disparities on bone repair [49–51].

Health disparities as defined by the National Institutes of Health as differences in morbidity, mortality, and access to health care among population groups defined by factors such as socioeconomic status, gender, residence, and race or ethnicity. Although racial-genetic differences can be dangerous and reinforcing of ethno-racial categories, it is vital that the factors that influence this dramatic difference be discerned. What appears to offer a stronger explanation for these ethnic health disparities is a model of socioeconomic inequality, which generates chronically stressful life conditions implicating one’s health [52,53]. Challenges and opportunities for significant progress are numerous. For example, consideration of sex-linked differences in MSC osteogenesis requires reconsidering basal media definition (now carbon-stripped), dosing and sequestration strategies for multiple forms of sex steroid hormones (e.g. estriol vs. estradiol), and consideration of passage number and bioinformatics strategies. Therefore, it is of paramount importance that our community considers patient characteristics in the fields of tissue engineering, regenerative medicine, immunology, and stem cell research as actionable, and reportable, experimental variables.

Conclusions

MC scaffolds provide a well-characterized platform inspired by the native bone tissue microenvironment. They increasingly allow for ex vivo studies to examine mechanisms of multicellular interactions, immunomodulation, and angiogenic activity essential for regenerative potency. They also provide motivation for a new generation of studies, enabled by advances in spatial-omics, synthetic biology, and stem cell engineering. However, it is essential these endeavors be designed from the outset to ensure we are responsibly understanding variability in order to effectively reduce health disparities.

Data Availability

No data were used for the research described in the article.