polymers Review Bone Matrix Non-Collagenous Proteins in Tissue Engineering: Creating New Bone by Mimicking the Extracellular Matrix Marta S. Carvalho 1,2,3,* , Joaquim M. S. Cabral 2,3 , Cláudia L. da Silva 2,3 and Deepak Vashishth 1,* 1 Center for Biotechnology and Interdisciplinary Studies, Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA 2 Department of Bioengineering and iBB—Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal; [email protected] (J.M.S.C.); [email protected] (C.L.d.S.) 3 Associate Laboratory i4HB—Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal * Correspondence: [email protected] (M.S.C.); [email protected] (D.V.) Abstract: Engineering biomaterials that mimic the extracellular matrix (ECM) of bone is of significant importance since most of the outstanding properties of the bone are due to matrix constitution. Bone ECM is composed of a mineral part comprising hydroxyapatite and of an organic part of primarily collagen with the rest consisting on non-collagenous proteins. Collagen has already been described as critical for bone tissue regeneration; however, little is known about the potential effect of non-collagenous proteins on osteogenic differentiation, even though these proteins were identified some decades ago. Aiming to engineer new bone tissue, peptide-incorporated biomimetic materials have been developed, presenting improved biomaterial performance. These promising results led to ongoing research focused on incorporating non-collagenous proteins from bone matrix to Citation: Carvalho, M.S.; Cabral, enhance the properties of the scaffolds namely in what concerns cell migration, proliferation, and J.M.S.; da Silva, C.L.; Vashishth, D. differentiation, with the ultimate goal of designing novel strategies that mimic the native bone ECM Bone Matrix Non-Collagenous for bone tissue engineering applications. Overall, this review will provide an overview of the several Proteins in Tissue Engineering: Creating New Bone by Mimicking the non-collagenous proteins present in bone ECM, their functionality and their recent applications in Extracellular Matrix. Polymers 2021, the bone tissue (including dental) engineering field. 13, 1095. https://doi.org/10.3390/ polym13071095 Keywords: non-collagenous proteins; extracellular matrix; bone tissue engineering; biomimetic scaf- folds Academic Editor: Alessandro Pistone Received: 28 February 2021 Accepted: 20 March 2021 1. Introduction Published: 30 March 2021 New promising solutions for bone tissue engineering have been developed over the last years following the dramatic increase of the number of bone-related medical conditions Publisher’s Note: MDPI stays neutral that require clinical interventions. In fact, each year, more than one million non-union with regard to jurisdictional claims in fractures are treated in the United States [1]. Moreover, 5–10% of the bone fractures that published maps and institutional affil- occur worldwide do not heal [1]. Besides bone fractures, bone tissue can also be damaged iations. by traumas, tumors, infections, or bone diseases. Furthermore, new strategies to engineer bone tissue are required as an alternative to the use of bone grafts, addressing the increasing worldwide incidence of bone disorders in an aging society severely impacted by obesity, lack of exercise, and with reducing healing capacity [2]. Even though bone tissue engi- Copyright: © 2021 by the authors. neering appears as a promising alternative, to date the gold-standard treatment for bone Licensee MDPI, Basel, Switzerland. regeneration still relies on bone grafts, autologous, allogenic, and xenogeneic grafts [1] This article is an open access article (Figure1). These approaches have some limitations and are not ideal for bone regenera- distributed under the terms and tion. Autografts have been applied since they can provide a matrix with osteogenic cells conditions of the Creative Commons and osteoinductive factors to support new bone growth, without having immunological Attribution (CC BY) license (https:// rejection and promote better osseointegration. Nonetheless, the availability of quality graft creativecommons.org/licenses/by/ material is limited and possible complications may occur, such as pain, infections, scarring, 4.0/). Polymers 2021, 13, 1095. https://doi.org/10.3390/polym13071095 https://www.mdpi.com/journal/polymers Polymers 2021, 13, 1095 2 of 33 and weakening of the donor bone. Moreover, there is a high morbidity associated with this procedure, since more than one surgery is needed [1,3]. Allografts, usually harvested from cadavers, have also some limitations, namely the higher risk of immunologic rejection and infection though it requires less procedures than autografts, minimizing the surgical time and accelerating the patient recovery [3]. As an alternative to allografts, xenografts consist of transplantation of bone tissue across species. The most common xenograft used in orthopedic surgery is bovine derived. Xenografts have some advantages compared to other grafts, such as being readily available due to the abundance of donor bone tissue and being less expensive than allografts [2,3]. In fact, commercially-available xenografts are approximately one-tenth the price of commercially-available allografts. Also, because of Polymers 2021, 13, x FOR PEER REVIEW 28 of 34 the extensive sterilization processes, their shelf life is generally long. However, xenografts present several challenges such as the risk of disease transmission and a higher risk of im- mune response of the host tissue compared to allografts [3]. Moreover, xenografts require intensive sterile processing, which can decrease their osteoinductive properties. FigureFigure 1. Comparison 1. Comparison between between autografts, autografts, allografts, allografts, and and xenografts: xenografts: Advantages Advantages and disadvantages. and disadvantages. Bone tissue engineering has the potential for solving these problems by combining different elements such as cells, molecules, and scaffolds. The standard tissue engineering approach uses a combination of growth factors, scaffolds and osteogenic cells (triangular Polymers 2021, 13, 1095 3 of 33 Polymers 2021, 13, x FOR PEER REVIEW 29 of 34 Bone tissue engineering has the potential for solving these problems by combining concept).different elementsHowever, such Giannoudis as cells, molecules,and colleagues and scaffolds.developed The and standard discussed tissue a new engineering concept, theapproach diamond uses concept, a combination in which of a growth fourth factors,element, scaffolds vascularization, and osteogenic should cellsbe also (triangular consid- eredconcept). as a contributor However, Giannoudisto bone healing. and colleaguesThus, the diamond developed concept and discussed in bone tissue a new engineer- concept, ingthe diamondcombines concept, four basic in whichelements a fourth [4]: (i) element, A biomaterial vascularization, with osteogenic should be ability also considered for bone formationas a contributor that acts to as bone a scaffold healing. for Thus, the other the diamond elements; concept (ii) osteogenic in bone cells tissue capable engineering to cre- atingcombines or inducing four basic new elements bone formation [4]: (i) A at biomaterial the defect site; with (iii) osteogenic osteoinductive ability formolecules bone forma- that triggertion that cells acts and as arecruit scaffold resident for the cells other to elements; form new (ii) functional osteogenic bone cells tissue; capable and to (iv) creating vascu- or larizationinducing newto support bone formation the viability at theof the defect defect site; site (iii) thus osteoinductive allowing the molecules diffusion that of oxygen trigger andcells nutrients and recruit to the resident defect cells region to form (Figure new 2). functional bone tissue; and (iv) vascularization to support the viability of the defect site thus allowing the diffusion of oxygen and nutrients to the defect region (Figure2). A B Biomolecules immobilized to enhance cell functions Figure 2. Bone tissue engineering strategies. (A) The bone tissue engineering paradigm highlights (1) Biomimetic scaffold, Figure(2) osteogenic 2. Bone cells,tissue (3) engineering osteoinductive strategies. molecules, (A) The and bone (4) tissue vascularization. engineering ( Bparadigm) Schematic highlights representation (1) Biomimetic of biomolecules scaffold, (2)immobilized osteogenic into cells, a porous (3) osteoinductive scaffold (left) molecules, and a functionalized and (4) vascularization. hydrogel with bioactive (B) Schematic peptides representation and cells incorporated of biomolecules (right). immobilized into a porous scaffold (left) and a functionalized hydrogel with bioactive peptides and cells incorporated (right). Several scaffolds have been developed for bone tissue engineering, including natural biomaterials (such as collagen, gelatin, and chitosan), ceramic implants (such as hydrox- yapatite),Several polymeric scaffolds synthetichave been materials developed (such for bone as polylactic tissue engineering, acid (PLA) including and polyglycolic natural biomaterialsacid (PGA)) (such and composite as collagen, scaffolds gelatin, [and5]. Even chitosan), though ceramic materials implants science (such technology as hydroxy- has apatite),resulted polymeric in clear