Abstract

Tissue engineering has become one of the most studied medical fields and appears to be promising for the regeneration of injured bone tissues. Even though the bone has self-remodeling properties, bone regeneration may be required in some cases. Current research concerns materials employed to develop biological scaffolds with improved features as well as complex preparation techniques. Several attempts have been made to achieve compatible and osteoconductive materials with good mechanical strength in order to provide structural support. The application of biomaterials and mesenchymal stem cells (MSCs) is a promising prospect for bone regeneration. Recently, various cells have been utilized alone or in combination with biomaterials to accelerate bone repair in vivo. However, the question of what cell source is the best for use in bone engineering remains open. This review focuses on studies that evaluated bone regeneration using biomaterials with MSCs. Different types of biomaterials for scaffold processing, ranging from natural and synthetic polymers to hybrid composites, are presented. These constructs demonstrated an enhanced ability to regenerate the bone in vivo using animal models. Additionally, future perspectives in tissue engineering, such as the MSC secretome, that is the conditioned medium (CM), and the extracellular vesicles (EVs), are also described in this review. This new approach has already shown promising results for bone tissue regeneration in experimental models.

Key words: tissue engineering, stem cells, materials, secretome

Streszczenie

Inżynieria tkankowa stała się jedną z najlepiej rozwijających się dziedzin medycyny i przypuszczalnie wysoce obiecującym rozwiązaniem w regeneracji uszkodzonych tkanek kostnych. Pomimo tego, że kość ma właściwości auto-modelujące, w niektórych przypadkach może być wymagana wspomagająca regeneracja kości. W obecnie prowadzonych badaniach w inżynierii tkankowej stosuje się materiały naturalne oraz syntetyczne do tworzenia rusztowań dla komórek z wykorzystaniem różnych technik ich przygotowania. Stosowane membrany wykazują strukturę zbliżoną do tkanki kostnej oraz dobrą wytrzymałość mechaniczną zapewniającą mechaniczne wsparcia dla komórek. Obiecujące wyniki w regeneracji kości uzyskano stosując membrany i mezenchymalne komórki macierzyste (MSCs). W inżynierii tkankowej wykorzystywane są komórki macierzyste różnego pochodzenia, które wprowadza się do miejsc ubytku bezpośrednio lub w połączeniu z materiałami w celu przyspieszenia naprawy kości in vivo. Brak jest odpowiedzi na pytanie, jakie źródło komórek macierzystych najlepiej nadaje się do wykorzystania w inżynierii kości. Niniejsza praca koncentruje się na badaniach oceniających regenerację kości przy użyciu różnych materiałów w połączeniu z MSCs. Przedstawiono różne rodzaje materiałów – od naturalnych i syntetycznych do hybrydowych polimerów stosowanych w przygotowaniu rusztowań dla komórek macierzystych. Omawiane rusztowania wykazywały wysoką zdolność do regeneracji kości w modelach in vivo. W tym artykule omówiono również perspektywy rozwoju inżynierii tkankowej opartej na połączeniu membran z czynnikami wydzielanymi przez komórki macierzyste, określanymi jako „sekretome”, czyli pożywkę kondycjonowaną (CM), oraz pozakomórkowe pęcherzyki (EVs). To nowe podejście metodyczne dało obiecujące wyniki w zakresie regeneracji tkanki kostnej w modelach eksperymentalnych.

Słowa kluczowe: komórki macierzyste, inżynieria tkankowa, materiały, czynniki wydzielnicze komórek macierzystych

Introduction

Millions of bone grafting surgical procedures for the partial excision of bone are performed worldwide.^{1, 2} In general, autograft transplantation and allograft transplantation are the standard clinical procedures.¹ However, they are associated with postoperative complications and availability problems.¹ In this context, tissue engineering is a potential alternative for tissue transplants and constitutes a novel approach in regenerative medicine that involves using natural and synthetic materials in combination with stem cells to repair the damaged areas.^{3, 4} The bone tissue engineering strategy consists of 3 essential components: scaffold, mesenchymal stem cells (MSCs) and bioactive growth factors. Mesenchymal stem cells are cultured on 2D or 3D scaffolds to induce the growth of new bone tissue through osteoinductivity.^{1, 3, 5} Studies have focused on the use of MSCs seeded on a scaffold due to their differentiation potential and paracrine/autocrine effects, which are important for tissue regeneration. The main source of cells used in tissue engineering is the bone marrow (BM).¹ The procedure of stem cell isolation from the BM is highly invasive and painful, which limits their application in tissue engineering, thus, new sources of MSCs are urgently needed. Adult stem cells such as adipose-derived stem cells (ASCs), umbilical cord blood mesenchymal stem cells (CB-MSCs) and oral mesenchymal stem cells (OMSCs) are among the candidates for bone tissue engineering applications.^{1, 3} The second component is the membrane used as a platform for MSCs. The scaffold can be a substrate that forms a fibrous network, which is a prerequisite for later bone formation.^{5, 6} Many parameters are involved in scaffold design that affects the mechanical properties and biological function of the scaffold while directly affecting the rate of bone regeneration.^{6, 7} The desirable parameters of a scaffold as a platform for MSCs are biodegradability, biocompatibility, imitation of the microenvironment, incorporation of different extracellular matrices (ECMs), stability, porosity, non-immunogenicity, interconnectivity, safety (low or no toxicity), and alignment.⁵ Designing scaffolds made from natural and synthetic components for bone engineering involves several parameters, such as physical and mechanical properties, chemical composition, and biological activities that affect scaffold properties and stem cell behavior.^{3, 5} The viability and differentiation of MSCs towards osteoblasts were found to depend on the intrinsic properties of the material and the interactions of specific chemical components of materials with stem cells.^{3, 5, 7} Several hybrid systems in the form of 2D and 3D natural and synthetic scaffolds have been fabricated and used for bone engineering; however, a good scaffold has not been developed yet.^{1, 4, 7, 8} Many papers discussed the advantages and drawbacks of different types of scaffolds with relevant examples of the impact of scaffold on stem cell biological behavior during stem cell differentiation and growth.^{3, 4, 5, 7, 9} Despite the advanced techniques and the variety of substrates used for the production of scaffolds, there is currently no construct with ideal parameters for tissue engineering. Consequently, one of the greatest challenges in bone tissue engineering is designing a scaffold with optimal architecture for cell growth and differentiation. This review summarizes the current developments in scaffold design as well as the effect of scaffolds on stem cell growth and the interaction between stem cells and materials in the context of bone engineering.

Selection and parameters of scaffolds used for bone tissue engineering

The selection of a suitable scaffold is crucial in bone engineering. Ideally, scaffolds should be similar to native tissue structure and degrade in a controlled manner consistent with the formation of new tissue.^{1, 2, 3, 4} The structure of a scaffold should allow the stem cells to release many particles affecting the surroundings of the damaged tissue.¹⁰ Conductivity, porosity, tortuosity, scaffold architecture, and financial cost should be considered for scaffold fabrication.1,4,7 The chemical content of the scaffold also has a major effect on osteogenic cell differentiation and should support the osteoinduction, osteoconduction and osteointegration of the stem cells seeded on it.^{11, 12} The components used in scaffold design can be classified into natural or synthetic.

Natural biomaterials

For scaffold design of natural biomaterials, different proteins such as collagen (Col), fibrin, gelatin, silk, and polysaccharides (agarose, alginate (Alg), hyaluronan, and chitosan), which are found in the ECM, have been used.^{1, 3, 5} These bioactive molecules usually contain sites for cellular adhesion, such as the arginyl–glycyl–aspartic acid (Arg–Gly–Asp) binding sequences, and can release non-toxic substances while degrading.⁹ Natural scaffolds are often more biocompatible than synthetic ones, and have the advantage of providing specific cell interactions.^{4, 8} However, the disadvantages of natural scaffolds include the difficulty in obtaining large amounts of the material, risk of transmitting pathogens and limited mechanical properties.⁹ Nonetheless, these proteins are very important for tissue structure and function, which justifies their choice in tissue engineering.⁵ Collagen is the crucial structural protein in human tissues that contains sites for cell adhesion. Moreover, Col builds the tissue basement membrane and has natural properties similar to those of soft human tissue.⁹ The main feature of Col is a unique structure rich in amino acids, such as glycine, proline and hydroxyproline.¹³ Collagen is non-toxic, non-immunogenic and expands when exposed to water in order to better fill the cavities in the damaged tissue and support the structural integrity of organs and tissues.¹¹ The disadvantages of Col as a natural biopolymer include a high probability of deformation and low mechanical stability.¹⁴ Collagens mixed with nano-inorganic materials are used to prepare scaffolds more widely than an ECM imitation of the bone in bone repair.¹⁵ Many attempts have been made to modify Col to achieve a good form for use in bone regeneration.¹⁵ Hyaluronic acid (HAc) is a natural polymer that consists of disaccharides, such as D-glucuronic and N-acetyl-D-glucosamine connected by β(1-4) and β(1-3) bonds within its polymeric structure.¹⁴ Hyaluronic acid is classified as a glycosaminoglycan. It has a highly consistent structure, except for deacetylated glucosamine residues, which occur occasionally.¹⁴ Molecular weight was found to play an important role in the biological, chemical, physical, and degradable properties of HAc.¹⁶ The pivotal physicochemical properties of HAc, such as biodegradability, biocompatibility, non-inflammatory, non-toxic and non-immunogenic behavior, allow for its wide application in drug delivery bioengineering and biomedicine, e.g., endoprosthesis of the joint fluid, wound dressings and polymeric scaffolds.^{16, 17} Chitosan is obtained from the N-deacetylation of chitin and consists of 2 D-glucosamine units: the deacetylated unit and the acetylated unit such as N-acetyl D-glucosamine. These units are randomly distributed within the polymer and linked by β-(1-4)-glycosidic bonds. Moreover, their derivatives showed several biological properties, including anti-inflammatory, antitumoral, antimicrobial, and antioxidant activities.^{18, 19} Chitosan is biocompatible and thermo-responsive, has excellent strength and stability, and is often being used as a gelling agent.¹⁸ Many tissues such as cartilage, bone, skin, and blood vessels have been regenerated by CS or CS in combination with ceramics or other biopolymers.¹⁸

Synthetic biomaterials

An alternative to natural materials is synthetic biomaterials for bone tissue engineering. Synthetic biomaterials include biodegradable polymers, such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly-ε-caprolactone (PCL), poly(ethylene glycol) (PEG), polyvinyl alcohol (PVA), polyurethane (PU), poly(lactic-co-glycolic acid) (PLGA), and ceramics-based biomaterials such as hydroxyapatite (HA) and bioactive glass. Scaffolds can be fabricated from these materials in controlled conditions to optimize the chemical and physical parameters of a scaffold.^{1, 3, 4, 5, 20} These materials have many advantages over their natural counterparts, including reproducibility and the possibility of controlling their mechanical properties. Likewise, the degradation rate can be controlled independently of shape.^{3, 5, 20, 21}

Chemical and physical modification of the polymer surfaces can increase their biological properties.⁵ Polymers derived from natural sources, especially those derived from the ECMs, are very good materials for tissue engineering due to their intrinsic bioactivity and self-assembling ability.⁴ A major drawback of natural polymers is the batch-to-batch variability, which makes it difficult to process at a huge scale because of changes in chemical composition and mechanical properties.⁶ Due to the lack of cell adhesion properties, many synthetic biomaterials must be chemically modified to allow stem cell adhesion and growth.^{5, 8, 9} Moreover, another disadvantage of polymers is their inadequate vascularization compared to natural bones.¹¹ Poly(lactic acid) is an aliphatic polyester and a hydrophobic polymer that naturally degrades through the process of hydrolysis: the ester bonds are broken down by water molecules, with the ester bonds determining the polymer structure.²² Poly(lactic acid) has good tensile strength and occurs in various forms, such as poly(D-lactic acid) (PDLA) and poly(L-lactic acid) (PLLA).^{21, 22} Unfortunately, PLA has some disadvantages, such as high fragility and lack of reactive side chain groups, which causes chemical inertness and low cell affinity due to its hydrophobicity.²³ Poly(lactic acid) has good potential for bone regeneration, but there are still certain problems, such as the mechanical modulus and the strength of bone scaffolds based on PLA, which is much weaker than cortical bone, particularly when PLA is produced with high porosity.²⁴ Poly-ε-caprolactone is a synthetic material with a melting temperature ranging between 59°C and 64°C (i.e., above body temperature) and a glass transition temperature of −60°C.²⁵ Poly-ε-caprolactone scaffolds have good elasticity and ductility, which shows that PCL has great potential for application in tissue engineering.²⁵ The presence of 5 hydrophobic –CH2 moieties in PCL repeating units makes it degrade the slowest among all the polyesters.²⁶ Poly-ε-caprolactone exhibits promising mechanical properties with good flexibility and substantial elongation, conducive to the construction of scaffolds for craniofacial bone repair.^{25, 26}

Bioceramics can be classified into 3 main types: bioinert (alumina and zirconia), bioactive (HA and Bioglass®) and biodegradable (calcium sulfate and tricalcium phosphate (TCP)).²⁷ Ceramics are bioinert, bioactive, fragile, bioresorbable, and characterized by good biocompatibility and high toughness. However, they are vulnerable to tensile stress and can be damaged due to high mechanical stress.⁵ Hydroxyapatite and TCP are widely used materials.^{1, 5} The presence of both, known as biphasic calcium phosphate (BCP), promotes the formation of apatite on the surface of the scaffold, which integrates with the host bone after in vivo implantation.^{1, 5} Hydroxyapatite has been used in tissue engineering because of its chemical similarity to the mineral component of bone tissue.²⁸ The size of the molecules of the compounds used to build the scaffolds is also an important parameter.¹¹ Scaffolds made of hydroxyapatite showed good stability in animal models and sustained their mechanical properties for a long period.⁴

Architecture of the scaffold

A different issue critical to scaffold success is the creation of a suitable microenvironment for the stem cells to differentiate into osteoblasts and form a new bone. Other problems concern the vascular supply upon bioimplant implantation or even the high density of seeded cells on the scaffold.⁴ In a porous scaffold, the density and size of pores affect cell migration and adherence to the scaffold, as well as nutrient and oxygen diffusion.^{4, 29} Too small pores may inhibit cell migration to the central layers of the scaffold, whereas the surface available for stem cell attachment in large pores may be limited. Moreover, the connection between the cells may not be enough to fill the gaps between the pores.^{3, 4, 7, 29} Tortuosity is another factor that affects mass transport, in turn affecting the migration of nutrients and cells within the scaffold structure.^{7, 30} Tortuosity refers to the path that the culture medium has to take through the pores to move from one point within the scaffold to another.^{7, 30} Surface roughness is a key parameter to take into account when designing scaffolds for tissue engineering. Roughness regulates the biological reaction of tissues in contact with the bioimplant. In vitro and in vivo studies showed that cellular morphology, proliferation rate and cell phenotype depend on the surface roughness of the material.^{1, 31} Technologies for scaffold preparation used in bone tissue engineering are very important because they affect cell behavior. The fabrication technique should be selected based on the architectural structure of the scaffold, which depends on the type of engineered tissue. Electrospinning is one of the new processing technologies to obtain suitable membranes for tissue engineering.^{7, 11} Freeze-drying is used to produce porous scaffolds through sublimation.¹¹ The main advantage of scaffold fabrication using the freeze-drying technique is that it does not require high temperatures.¹¹ Gas foaming is a technique that can produce highly porous scaffolds using high-pressure carbon dioxide gas.²² Additive manufacturing includes selective laser sintering (SLS) and 3D printing, which enables independent control over pore sizes, shapes, porosity, and roughness during scaffold designing.³² This technology allows different types of materials to be used, ranging from ceramics and polymers to hybrid composites. Moreover, additive manufacturing allows scaffolds to be designed for a wide range of tissue applications.^{32, 33, 34, 35, 36}

Source of stem cells used in bone engineering

Mesenchymal stem cells obtained from different parts of the body, such as the BM (BM-MSCs), fat cells (ASCs) or dental pulp (dental pulp stem cells (DPSCs)), show the capability to differentiate into various types of cells and are the closest to the stem cells involved in the reconstruction of bone damage.^{10, 11, 12, 37} However, as presented in Table 1, the aforementioned subpopulations of cells used in bone defect repair have both advantages and disadvantages.^{10, 12, 37} The BM-MSCs have a high osteogenic potential and may possess proangiogenic paracrine effects involved in vascular reconstruction.^{32, 37} The BM-MSCs are recognized based on specific criteria: fibroblastic morphology, the ability to adhere and form clusters, and the potential to differentiate into 3 lineages (endoderm, mesoderm and ectoderm).³² Many successful bone healing procedures have been performed using BM-MSCs (through direct injection or in combination with 3D scaffolds) in both calvarial and long bones in different animal models.³² The DPSCs are stem cells harvested from the dental tissue of both adults and children.^{10, 12} The DPSCs easily differentiate into osteoblasts (Figure 1).^{10, 12} In recent years, DPSCs have been considered applicable in bone engineering. These cells are osteogenic-specific markers (e.g., osteopontin, osterix and osteocalcin) and can form bone-like nodules in vitro.³⁷ The ASCs have been targeted as useful cells for bone engineering due to their ability to differentiate into several different types of cell lineages, ease of accessibility, immunogenic capabilities, and stability in long-term cell cultures.³⁸ Contrary to BM-MSCs, ASCs do not lead as quickly to senescence and easily differentiate into osteoblasts.¹² Moreover, they have high vascularization properties, which benefit bone reconstruction.¹² The ASCs are also a rich source of biologically active factors that are secreted via exosomes, which can help in the signaling pathways that differentiate cells into bone.³⁹

Impact of different scaffolds on the osteogenic potential of stem cells in bone engineering

Until now, 235 ongoing clinical studies involving MSCs have been used for several medical conditions (https://www.clinicaltrials.gov/). However, some limitations of MSC therapy have been observed, particularly the short MSC survival time after injection, the right doses and the best route of administration are unclear.⁴⁰ To avoid such complications, MSC-based tissue engineering approaches have been applied.⁴⁰ In bone engineering, scaffolds work as a temporary supporting platform to (a) ensure that the tissue regeneration area is suitable for bone formation and remodeling, (b) provide and maintain mechanical functionality during tissue regeneration, and (c) facilitate growth and angiogenesis during tissue regeneration.²⁹ The mechanism of bone regeneration induced by scaffolds is determined through the scaffold’s chemical and biological environment, where the stem cells differentiate and grow into osteoblasts.²⁹ However, the mechanisms by which scaffold-induced bone regeneration takes place are not fully described, and many problems must still be explained and solved. For example, one problem is the lack of a functional vascular supply after MSC implantation, even for scaffolds possessing good biocompatibility with regenerated tissue.^{3, 5, 7} A crucial and challenging requirement that an appropriately designed scaffold must meet is mimicking the dynamic nature of the native tissue.⁴ Scaffolds for bone tissue engineering should imitate the natural matrix. Natural and synthetic polymers, ceramics, and composites are the most frequently used materials.^{3, 4, 5, 7} Among natural scaffolds, the Col sponge is the most commonly used material in bone repair because Col is the important component of the ECM and widely occurs in the bone.^{8, 9, 41} Great efforts have been made to design Col-based scaffolds that exhibit similarity to the native environment.⁴² Collagen is frequently added to synthetic scaffolds to increase their bioactivity and allow stem cells to attach to the scaffold surface.⁹ Modification of gelatin methacryloyl (GelMa) can serve as a valuable membrane for MSCs in bone tissue engineering.⁹ Recently, heparin-modified mineralized Col scaffolds with seeded BM-MSCs have been successfully used in bone repair because of naturally occurring bioactive factor mixtures promoting cell proliferation, migration and vascularization.⁴³ Furthermore, Wang et al. found that Alg–Col composite hydrogels seeded with BM-MSCs promoted proliferation and osteogenic differentiation as well as enhanced new bone formation in a critical-sized calvarial defect animal model.⁴⁴

Hyaluronan scaffolds combined with MSCs have been used in cartilage and bone tissue engineering.⁴⁵ Human DPSCs (hDPSCs) were seeded on a hyaluronan-based biomaterial and tested for bone repair.³⁴ The Woodhouse group identified that using adipose substitutes from stem cells in bone engineering demonstrated better effect in the new bone tissue and blood vessel formation than using only the scaffold.⁴⁶ New approaches in tissue engineering are based on hydrogels/scaffolds combining materials, small molecules and stem cells for bone engineering.³⁶ Liu et al. modified CS hydrogels by incorporating catechol (CA) and zeolitic imidazolate framework-8 nanoparticles (ZIF-8 NP) to enhance the adhesive properties of the hydrogel. The modified hydrogels exhibited antibacterial properties and induced new vessel formation and osteogenesis.³³ Interesting data were reported by Sancilio et al., who combined hDPSCs with an Alg/HA scaffold and found that the scaffold enhanced hDPSC proliferation and differentiation towards osteoblasts and was capable of promoting successful tissue regeneration and calcium deposition, as well as sustaining natural bone formation.³⁵ Fouad et al. observed that the porous calcium sulfate–HA (CS–HA) coating of the scaffold with seeded BM-MSCs not only improved the attachment and proliferation of BM-MSCs but also enhanced the process of osteointegration in the therapy of critical-sized calvarial bone defects.⁴⁷ Another report found that a highly interconnected porous Alg/HA scaffold coated with lactose-modified CS (CTL) and cultured with hDPSCs increases the proliferation rate and osteogenic differentiation of hDPSCs.⁴⁸ Gutiérrez-Quintero et al. demonstrated that a HA matrix and a PLGA (HA/PLGA) scaffold seeded with hDPSCs induced angiogenesis and bone regeneration in rabbit bilateral mandibular critical-sized defects more effectively than a scaffold without hDPSCs.⁴⁹ Tschon et al. showed that a scaffold with zoledronate and HA nanocrystals (HAZOL) combined with human BM-MSCs (hBM-MSCs) not only enhanced stem cell adherence and movement into the scaffold but also increased the expression of bone-related proteins.⁵⁰ Some reports showed that a HA–Col scaffold created a microenvironment that was conducive to the osteogenic differentiation of dental stem cells and may be the optimal design for maxillofacial and alveolar bone regeneration.⁵¹ Promising results were observed when BM-MSCs were seeded on a HA–Col scaffold.⁵¹ In this study, a commercially available HA–Col scaffold was used to culture DPSCs.⁵¹ Furthermore, this bioactive material induced osteogenic differentiation of hDPSCs and may be used for maxillofacial and alveolar bone regeneration.⁵¹ When BM-MSCs were seeded on an HA–Col scaffold, no biological toxicity was observed. Moreover, the HA–Col scaffold promoted BM-MSC adhesion, proliferation and differentiation into osteoblasts.⁵² Alginate and nano-sized HA (nHA) scaffolds (Alg/nHA) were also tested for bone regeneration.³⁵ Dental pulp stem cells seeded onto a scaffold based on Alg and nHA expressed osteogenic differentiation-related markers and promoted calcium deposition. Moreover, it can efficiently sustain natural bone regeneration.⁵³ Lorusso et al. found that the synthetic scaffolds in combination with DPSCs showed high ability to support both osteoblast and osteoclast generation in new bone formation in an animal model, and could be useful for bone regeneration procedures.⁵⁴ The authors described different scaffolds such as hydrogels, nanofibers, βTCP, and HA combined with the DPSCs for bone regeneration. Furthermore, they identified that the advantage posed by scaffold use is determined by the physical space-maintaining capability of the regenerative area, the three-dimensional structure of the scaffold and bioactive factor, and the cellular osteogenic response of the host.^{44, 54} Interesting data were presented when bioceramics, such as HA and TCP, were modified to increase the osteogenic potential of stem cells within 3D scaffolds.⁵⁵ The nHA features of the scaffold generate better cellular responses compared to micron-sized particles (mHA).⁵⁵ The nanophase of the ceramics revealed the strong adsorption of vitronectin, which promotes osteoblast migration, adhesion and enhanced osteoclast-like cell function compared to conventional HA.¹

Several authors have developed scaffolds combining calcium phosphate and HA with Col, Alg and CS.⁵⁶ In this context, Col has been extensively used with TCP and HA for both in vitro and in vivo studies, supporting both woven and lamellar bone formation and growth.^{54, 57} In an experimental study investigating a hybrid scaffold consisting of type I Col and HA, the adhesion and proliferation of MSCs and human periodontal ligament stem cells increased.²² The proposed scaffold was a composite structure made with ceramic (HA, HA-magnesium (Mg) and HA-silicon (Si)) and Col, and demonstrated better mechanical stability and improved MSC proliferation activity.⁵⁸ Marine spongin combined with HA accelerated material degradation and improved new bone formation.⁵⁹ In mouse critical-sized defects, a bioceramic material composed of dicalcium phosphate and HA showed a better regenerative effect than an implant made of BCP with Col.⁶⁰ This type of bioimplant revealed other advantages, such as early resorption of material and high bone formation.⁶⁰ In the treatment of a large critical-sized bone defect in rabbits, the addition of MSCs on silica-coated calcium–HA scaffolds induced higher osteogenesis than that observed with the scaffold alone.⁴⁶ When bioactive factors were introduced to the scaffold–MSC construct, the bone healing process was increased.⁴⁶ The use of the composite biomaterial poly(3-hydroxybutyrate)/HA/Alg with the addition of MSCs enhanced the regeneration process and facilitated bone formation in a critical-sized defect about fourfold.⁶¹ Bernardo et al. fabricated 3D-printed porous scaffolds composed of PLA/HA with loaded BM-MSCs for bone engineering. The authors showed that pure PLA scaffolds moderately induced an osteoconductive effect, whereas PLA/HA scaffolds efficiently enhanced osteogenic differentiation of MSCs even in the absence of any typical osteogenic stimuli.⁶² In the author’s opinion, 3D-printed PLA scaffolds supplemented by high concentrations of HA are most suitable for applications in bone tissue engineering.⁶² Comparable data were also obtained by other researchers for scaffolds fabricated using PLLA with different concentrations of nHA. These scaffolds were tested to reveal their impact on human ASCs (hASCs), morphology, proliferation rate, and adhesion.⁶³ Poly(L-lactic acid) functionalized with 10% of nHA positively influenced hASC biological features such as attachment and proliferation. Furthermore, an nHA/PLLA scaffold might be considered in bone tissue engineering.⁶³ Another study found that HA incorporated on PCL-based scaffolds showed good cytocompatibility and high osteogenic potential of hDPSCs, and PCL/HA scaffolds might be used for bone tissue engineering.⁶⁴ Moreover, another study reported that 21 days after CB-MSCs were seeded onto a scaffold composed of Geistlich Bio-Oss® transplantation, significant bone repair was found in a cranial defect.⁶⁵ Synthetic polymers include PLA and PGA, which exhibit good mechanical properties of the fibre scaffold and suitable porosity, tortuosity and roughness, are non-inflammatory, biodegrade, and can support stem cell adhesion to the scaffold.¹¹ A nanofibrous scaffold composed of PCL and Mg oxide (MgO) nanoparticles was used for bone tissue engineering.⁶⁶ The MgO nanoparticles added into PCL nanofibers improved the mechano-chemical properties and enhanced cell adhesion and viability of the seeded cells.⁶⁶ Before MSC-based applications are adopted, several challenges associated with cell therapy, such as standardization and quality of stem cell culture, regulatory approval, and the sources of stem cells, should be further investigated. Recently, to overcome the abovementioned limitations associated with stem cell therapy, research has focused on the approaches based on the MSC-derived secretome, such as the use of extracellular vesicles (EVs) and the cytokines and growth factors released by MSCs. Although promising valuable data have been reported, tissue engineering has faced problems related to the standardization and validation of the manufacturing processes.¹²

Scaffold modification using the secretome in bone engineering

Stem cells have a paracrine effect and release a wide range of macromolecules to the extracellular area as the conditioned medium, including proteins, lipids and nucleic acids, especially RNA.^{10, 12, 67, 68, 69, 70} Moreover, MSCs secrete EVs in the form of microvesicles (MVs), exosomes (EXs), apoptotic bodies, and microRNA.^{37, 69} Extracellular vesicles are important mediators of intracellular communication that enable the transmission of biological signals between cells.^{37, 71} As presented in Table 2, there are many reports examining bone defect repair using an MSC-derived secretome combined with a scaffold. Exosomes derived from hASCs were immobilized on polydopamine (pDA)-coated PLGA (PLGA/pDA) scaffolds. Then, the scaffolds were inserted into critical-sized calvarial defects in mice. The results demonstrated that the EXs enhanced the migration, proliferation and osteogenic differentiation of hBM-MSCs and promoted bone regeneration in the calvarial defects in an in vitro model.⁷² Khojasteh et al. compared the ability to repair mandibular bone defects in dogs using MSCs and endothelial progenitor cells (EPCs) seeded on βTCP scaffolds coated with PLGA microspheres that partially released the vascular endothelial growth factor (VEGF).⁷³ Bone formation was found to be most effective in the MSC/VEGF scaffold and then in the MSC/EPC/VEGF scaffold.⁷³ An experimental study presented by Liu et al. showed that the human periodontal ligament cells (hPDLCs) co-cultured with BM-MSC-derived small EVs (BMSC-sEVs) increased the osteogenic potential of hPDLCs.⁷⁴ Another study demonstrated that EXs obtained from MSCs and loaded onto a Col sponge increased new bone formation in a rat periodontal defect model.⁷⁵ Takeuchi et al. showed that the scaffold-MSCs-EX construct enhanced osteogenesis and the accumulation of vascular endothelial cells in a rat calvarial bone defect model.⁷⁶ The results obtained by Hiraki et al. proved that the bone regeneration of stem cells derived from human exfoliated deciduous teeth (SHED) and conditioned medium (CM) was associated with angiogenesis and osteogenesis in a mouse calvarial bone defect model.⁷⁷ The CM collected from cultured hBM-MSCs and human periodontal ligament fibroblasts (HPLFs) under cyclic stretching was implanted on a Col sponge to repair mouse calvarial bone defects.⁷⁸ The authors suggested that this construct was involved in bone regeneration and angiogenesis.⁷⁸ The CM obtained from periodontal ligament stem cells (PDLSCs) and gingival MSCs (GMSCs) and then used for periodontal defect regeneration demonstrated the regenerative potential of these cells.⁷⁹ Another study evaluated the regenerative ability of human PDLSCs (hPDLSCs) and GMSCs (hGMSCs) and their secretome. Moreover, CM and EVs seeded on Col or polylactide membranes in a rat calvarial defect model showed that the secretome modified and accelerated bone remodeling, but with different kinetics of mineralization.⁷⁰

Future prospects

A major challenge in bone engineering is using suitable MSCs and materials for bone repair. There are many different biomaterials used in combination with stem cells that have been used for bone reconstruction. In order to mimic the native ECM for bone tissue formation, further efforts are required to improve the interaction between stem cells and the scaffold. Attention should be given to a proper understanding of the kinetics of growth factors released by MSCs during bone regeneration. The construction of an optimal scaffold for bone tissue engineering is an important task because the scaffold must induce the interaction between MSCs and enhance osteogenic differentiation. Overall, a profound scientific knowledge of different types of scaffolds combined with different stem cells is necessary for future clinical usage, which might be important in entirely new applications.