Abstract

2.1. Biological factors

One of the main considerations for engineering a scaffold is to decide whether the scaffold is supposed to be a vehicle to deliver cells, growth factors, and biological factors or whether the scaffold alone will promote the tissue regeneration process. It is essential to consider the clinical demands when designing delivery-based scaffolds. To regenerate functional tissue, a similar cellular environment needs to be created for the cells [136]. Several studies reported the efficacy of bioactive materials for tissue regeneration and host integration. The biological factors can be incorporated into the engineered scaffolds through several physical and chemical techniques. They can be blended/encapsulated into the scaffold adsorbed/bound to the surface, and covalently conjugated to it.

In this section, the importance and critical requirements of biological factors for tissue regeneration, such as growth factors, and cell-based therapy, will be discussed.

2.1.1. Growth factors

Studies reported that the incorporation of growth factors could promote the therapeutic effects of the scaffolds [[137], [138], [139], [140], [141]]. The tissue regeneration process is regulated by complex signaling networks, including different growth factors, cytokines, and chemokines. Among them, some factors showed a significant role in regenerative engineering, including the transforming growth factor-β (TGF-β) [[85], [92], [130]], fibroblast growth factor (FGF) [91,120], hepatocyte growth factor (HGF) [[113], [129], [134]], vascular endothelial growth factor (VEGF) [130,120], insulin-like growth factor (IGF) [114], platelet-derived growth factor (PDGF) [105], nerve growth factor (NGF) [142,125], connective tissue growth factor (CTGF) [143,144], and interleukins (ILs) [98].

Besides the regenerative ability of growth factors, some of the critical parameters need to be controlled, such as the type of growth factor, dosage, and release profile at the repair site (Fig. 3).

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Fig. 3

It is essential to select the appropriate growth factors for a specific tissue. For example, angiogenesis is mainly regulated by the tissue oxygen level and tissue growth factors like hypoxic and chemokines [145,146]. The type of growth factor has a critical role during the tissue regenerative process. Several studies demonstrated the efficacy of using growth factors such as connective tissue growth factor (CTGF), TGF, and stromal-derived factor (SDF) to promote cell activity and angiogenesis In particular, a number of studies have highlighted the significant effects of CTGFs on endothelial cell proliferation, adhesion, migration, and vascularization [147]. Research showed a promoted viability of the endothelial cells in CTGFs-loaded scaffolds compared with scaffolds alone [143,144].

The osteoinductive effects of IGF-1, EGF, and VEGF on human Mesenchymal Stem Cells (hMSCs) by increasing the alkaline phosphatase (ALP) activity and mineralization have been shown in vitro [148]. In addition, these growth factors can show significant effects on other tissues. For example, IGF-1 has a significant role in osteogenesis, cardiogenesis, and tenogenesis [114,90]. Rosellini et al. formulated injectable microspheres using a combination of two natural polymers (gellan and gelatin) for cardiac repair. The microspheres were produced via water-in-oil emulsion and were further functionalized by loading with IGF-1. In vitro cell culture experiments demonstrated that these microspheres facilitated the adhesion of rat cardiac progenitor cells and the formation of clusters, indicating promising attributes for potential utilization as injectable scaffolds in cardiac tissue engineering endeavors [114].

Moreover, studies suggested the regenerative potential of multiple growth factors for tissue regeneration [130,120,129,[88], [131], [149]]. Ogino et al. investigated the efficacy of the dual-controlled release of hepatocyte growth factor (HGF) and the basic FGF (bFGF) infused with a collagen/gelatin scaffold using a mouse model of skin defects. Based on the results, the neo-epithelium length and wound closure were improved in the HGF-low dosage group compared with the normal saline solution group one week after the surgery. Two weeks after surgery, the bFGF and HGF + bFGF groups promoted wound closure, dermis-like tissue formation, neo-epithelium length, and newly formed capillary formation compared with the normal saline solution and HGF-high dosage groups. Also, the results showed the significant formation of the newly formed capillary in the HGF + bFGF group compared with the bFGF group [129].

The appropriate dosage and the release profile of the growth factors in the body are other important factors in controlling tissue regeneration during angiogenesis, myogenesis, osteogenesis, chondrogenesis, and neurogenesis [129,150]. For instance, appropriate dosages of BMP-2 and VEGF can change their efficacy during osteogenesis and angiogenesis [150]. Studies showed that using a smaller dosage of BMP-2 to induce osteoinductivity recruits stem cells via chemotaxis, while the higher dosage in the range of microgram has a differentiation stimuli role that has a direct effect on endochondral ossification [150]. Besides the dose dependency, the release profile of the growth factors is a major concern in tissue regeneration. Lipner et al. demonstrated that long-time presence and sustained release of BMP-2 in ASCs-seeded scaffold reduced the mechanical properties of the tendon-bone interface and caused bone resorption [81].

It has also been revealed that functional blood vessels can be regenerated through the slow and sustained release of VEGF, while an uncontrolled release showed a reverse effect [150].

Despite all the conducted studies to improve the efficacy of scaffolds by growth factors, the accessibility and cost of growth factors are still controversial and need to be addressed.

2.2. Cell-based therapy

Cell-based therapies have been considered a promising strategy for tissue regeneration. The therapeutic benefits of cell therapy have been demonstrated in various organs and tissues, such as the heart [[111], [151], [152], [153]], lungs [[154], [155], [156], [157]], intestine [[158], [159], [160]], liver [161], and the musculoskeletal system [80,162]. One of the critical challenges in these strategies is to choose the right cell source among autologous, allogeneic, or xenogeneic cells. Although allogeneic or xenogeneic are cost-effective and more efficient for the fabrication of an off-the-shelf scaffold, using autologous cells avoids immune rejection and consequently the need for immunosuppressive medicine. To address the current challenges in cell-based therapies, stem cells have attracted attention to be considered the primary cell source.

Pumberger et al. reported the regenerative ability of the rat bone marrow-derived MSCs (BM-hMSCs) for muscle regeneration in a rat model of traumas to the left soleus muscle [110]. Bone marrows were isolated from rat tibia and seeded onto the porous alginate scaffold fabricated by lyophilization with pore sizes ranging in 70–150 μm. The porous scaffold was utilized as the cell delivery vehicle to ensure the local delivery of hMSCs at the injury site. Based on the results, muscle treatment with hMSCs improved muscle fiber density and the number of regenerated fibers compared with control groups without cells at 4 weeks post-implantation [110].

Several studies show that the therapeutic effects of stem cells could be attributed to their secretome. For example, it has been reported that the therapeutic reason for cell-based strategy for neurological and brain disorders, such as multiple sclerosis (MS) and Parkinson's disease (PD), is attributed to the secretome secretion of stem cells [[124], [163], [164], [165], [166], [167]].

Also, it has been shown that MSCs can significantly reduce muscle degeneration in tendon-muscle injuries by promoting cell proliferation, reducing inflammation, and modulating the immune system [162]. Flück et al. examined the ability of injected autologous MSCs to prevent muscle degeneration via the stimulation of myogenesis and suppressing adipogenesis in the ISP muscle following delayed tendon repair. As the result, they observed that injecting autologous MSCs into degenerated rotator cuff muscles could stop the conversion of muscle to fat during the recuperation from tendon repair. This preservation of fat-free mass was achieved through extracellular reactions, which halted adjuvant-induced muscle fiber hypertrophy [162].

The potential of several synthetic and natural biomaterial-based scaffolds has been investigated as secretome delivery vehicles [80,[96], [97], [168], [169]]. Chen et al. have found that secretome of human bone marrow-derived stem cells (hBMSC) could improve tendon–bone healing through regulating macrophage polarization in a rat model of rotator cuff repair which was relied on its immune-regulative property [80]. In another study, Diomede et al. reported the beneficial effects of 3D-printed PLA and polyethyleneimine (PEI) scaffold incorporated with human gingival stem cell-derived extracellular vesicles on bone tissue regeneration in a rat calvarial bone defect [97]. The results demonstrated new bone formation, vascularization, and the potential of the construct to repair the defect [97].

Moreover, studies have also reported the efficacy of secretome for soft tissue regeneration. Recently Luo et al. investigated the therapeutic potential of the secreted secretome from the human bone marrow-derived mesenchymal stem cell in the mice model of myocardial infarction [111]. Injection of two-layer microparticles, including MSC-conditioned media/PLGA as the core and MSC cell membrane as the shell in the mice model with acute myocardial infarction demonstrated enhanced angiogenesis and myocardial functions [111].

Despite all promising outcomes, some challenges need to be addressed to translate cell-free-based therapies to the clinic. There is a need to investigate the ability to scale up the manufacture of this strategy at a commercial scale. Moreover,the standard procedures for cell isolation, culture, and purifying need to be developed.

2.3. Topographical factors

The surface topography of the engineered scaffold is another critical factor that can modulate the scaffold's biological response and clinical success. The topography of implant materials plays an important role in directing stem cell fate. In 1996, Recum et al. initially defined topography as the morphology of the surface and categorized it to surface roughness and texture [170]. Random size and distribution of surface hills and pits change the surface topography through roughness. However, changing the surface morphology through grooves, pores, ridges, porosity, and other configurations, can create surface texture [170].

Studies reported the roughness quantifications by measuring the protrusions or depressions at the surface [[101], [109], [171], [172], [173]]. The surface roughness can have significant effects on cell adhesion, migration, proliferation, and differentiation. For example, the microscale surface roughness has been recognized to alter the osteogenesis of stem cells [101,109]. By optimizing the roughness scale of the material surface to roughness (Ra) ≈ 0.77–1.09 μm, bone formation can be highly induced [101].

Besides different types of surface configurations, the orientation of surface texture can also be categorized as isotropic (no orientation, for example, randomly distributed fibers) or anisotropic (a clear orientation such as ridges, aligned fibers, and grooves). The anisotropic surface has been studied in terms of a tool to direct cell alignment, which often influences stem cell fate. The isotropic surface is not determined to influence cell alignment; instead, it is proved to control cell function. The surface texture can be created as either an anisotropic or an isotropic surface and modulate stem cell differentiation to target phenotype. Besides, it has been reported that the distribution of topographical features also influences cell response [[101], [109], [172], [173], [174]]. In this section, the most common types of surface texture that have been shown to affect cellular behavior in vitro and tissue response in vivo will be discussed.

3.1. Structure alignment

Tissues with highly aligned structures such as tendons, ligaments, skeletal, cardiac, and blood vessels show the alignment and migration of the cells along the direction of collagen fibers [1,23,172,174,183]. Studies demonstrated that the aligned topography of the engineered scaffolds can significantly induce cellular elongation, alignment, and differentiation and guide the cell migration along the direction of the scaffold [1,23,85,172,173,174,184,108]. The scaffold can be fabricated with highly aligned structures through topographical patterns such as ridges/grooves, wrinkles, pillars, and fibers in nano- or micro-scale [172]. Based on the literature, the micro-scale crinkles show more effects on cell attachment and migration, while the nano-scale crinkles significantly induce cell alignment. Moreover, it has been demonstrated that cell alignment can be affected by changing the width, depth, and geometries of grooves, pillars, and fibers [184,185,186].

The efficacy of multilayered scaffolds composed of highly aligned poly(l-lactic acid) (PLLA) nanofiber yarns was investigated by Li et al. [174]. According to the results, the engineered scaffolds with topographic factors showed higher mechanical properties, promoted H9C2 cell proliferation, and guided the cell orientation. Also, the controllable pore structure of the scaffolds allowed the cell to penetrate through the thickness of the 3D scaffold and distribute uniformly in each layer [174].

Liu et al. investigated the efficacy of controllable surface topography by fabricating a core-shell fibrous scaffold [172]. The scaffold was fabricated by electrospinning polycaprolactone (PCL) nanofibers onto polyglycolic acid (PGA) microfibers. Based on the results, A-PCLs group (a shell of aligned PCL nanofibers on the core of PGA yarn) with appropriate surface topography of PCL nanofibers and mechanical properties of PGA microfibers showed higher structural properties and promoted the adhesion and proliferation of BALB/3T3 (mouse embryonic fibroblast cell line). The cells presented an irregular oval form on the R-PCL scaffolds while significantly higher aspect ratio, attachment, and proliferation were observed along the A-PCLs [172].

The aligned structure as a topographical cue can be applied to the scaffold combined with other stimulation factors such as electroconductive biomaterials. Roshanbinfar et al. designed a composite fibrous mesh intended to mimic the cardiac ECM in microstructure and electrical conductivity. To achieve this goal, they fabricated electrospun fiber mats using collagen, hyaluronic acid and PANi as protein, polysaccharide, and a synthetic polymer respectively, at varying concentrations. These fiber mats demonstrated electrical conductivity similar to and superior mechanical properties compared to the native human myocardium. Analysis of cell-matrix interaction using postnatal rat cardiomyocytes indicates that the fiber mats are non-cytotoxic, facilitating cell attachment and contraction. Their findings proved the capability to tune various characteristics of the fiber mats, such as morphology, fiber alignment and diameter, mechanical properties, and electrical conductivity, by adjusting the composition of the fiber mats [108]. The significant effects of fiber alignment have also been reported on human adipose-derived mesenchymal stem cells (HADMSC) and human tenocytes (HT) differentiation and tendon-specific gene expression [85].

3.2. Electrical stimulation

External electrical stimulation is an approach that induces an electrical charge in cells to mimic the natural endogenous electrical potential [188]. External electrical stimulation has extensively been used in both clinics and research labs to treat neuronal diseases, muscular diseases, and wound healing [5]. Several studies investigated the effects of external electrical stimulation on different cell types such as, mesenchymal stem cells (MSC), myoblasts, fibroblasts, osteoblasts, and neural cells [29,67,69,139,[100], [103], [117], [126], [127], [128], [132], [189]]. Their results showed the regenerative effects of electrical stimulation on cell adhesion, proliferation, differentiation, elongation, and migration.

Tissues and organs in the body show different electrical resistivity due to the differences in tissue composition, tissue type, tissue density, cell membrane permeability, and electrolyte content. Owing to these variations in electrical resistivity, the method by which the electrical stimulation is applied must be optimized and adjusted to achieve the desired therapeutic effects [190]. There are various types of electrical stimulation strategies. According to the literature, electrical stimulation strategies can be divided into three categories, including direct current/alternating current electric field (DC/AC EF), capacitive coupling electric field (CCEF), and inductive coupling electric field (ICEF) [190]. Various cell types have been studied and found to be responsive to these ES methods under different experimental settings [29,67,69,139,[100], [103], [117], [126], [127], [128], [132], [189]].

Electrical stimulation is one of the promising approaches for cardiac regeneration to promote the differentiation of cardiac myocytes and induce contractions [29,139,117]. Studies evaluated the efficacy of external electrical stimulation alone or in combination with electroactive materials [29,67,69,[126], [127], [128],100]. For example, Zhao et al. reported the regenerative potential of aligned and random rGO/silk fibroin nanofibrous scaffolds with and without external electrical stimulation for cardiac regeneration [29]. The rGO/silk scaffold showed a high conductivity while maintaining the topological factors and flexibility of nanofibrous scaffolds. Upon seeding the scaffolds with rat cardiomyocytes and applying external electrical stimulation (1Hz, 5 V/cm for 4 days), the rGO/silk scaffold with aligned orientation promoted cell spreading, cardiac-specific protein expression, the formation of sarcomeric structure, the formation of cell-cell gap junctions, and spontaneous beating of regenerated cardiac tissues. Using the rGO layer enhanced the effect of electrical stimulation on cardiac tissues [29].

3.3. Mechanical stimulation

Mechanical stimulation as a biophysical cue has been applied to 3D engineered scaffolds to enhance, regulate, and accelerate the regeneration of functional tissue. Its role is to stimulate the synthesis of ECM, guide cell differentiation, enhance biomimetic construct organization, and improve the function of cells or tissues. This approach is one of the most effective factors for engineering a functional skeletal tissue, including bone [25], cartilage [191], ligament, tendon [85,83], and skeletal muscle [192] that significantly affects the development, rehabilitation, and healing of the tissues. It has been reported that mechanical stimulation induces MSCs differentiation even without biological factors [25,193,194].

Applying mechanical loading is a promising strategy to promote cartilage regeneration by improving chondrocytes' metabolic activity, composition, and biomechanical properties. Ouyang et al. reported the efficacy of dynamic tensile mechanical stimulation on the co-cultured rabbit chondrocytes and bone-derived MSCs for cartilage regeneration [191]. The results suggested the regenerative ability of this combination to promote cell proliferation and cartilage phenotype. The significance of mechanical stimulation has also been reported for skeletal muscle regeneration by providing similar mechanical loads to those seen in the body [107]. The study exhibited the effects of mechanical stimulation on cell proliferation, myofiber organization, myofiber diameter, ECM composition, and muscle elasticity [107].

The potential of mechanical stimulation to induce differentiation of stem cells and stimulate tendon/ligament regeneration has also been reported [85,82,83]. The use of mechanical stimulation aligns the cells along with the force direction resulting in the generation of aligned collagen fibers and upregulation of specific genes (Collagen I, Collagen III, tenascin C). Wu et al. reported the efficacy of dynamic stretch on human adipose-derived mesenchymal stem cells (HADMSC)/Human tenocytes (HT)/Human umbilical vein endothelial cells (HUVEC) tri-culture for promoting collagen secretion and tenogenic differentiation [85]. HADMSC/HT/HUVEC were seeded on the woven fabric scaffold and cultured under both static and dynamic environments for two weeks. The in vitro results showed the effects of the dynamic environment on enhancing the expression of collagen and tenogenic differentiation markers (SCX, TNC, COL1, COL3, TNMD, and VEGFA genes) compared to the static condition [85].

3.4. Morphological factors

The final step for designing scaffolds is to define the required fabrication technique. The morphology of the engineered scaffold is a critical consideration that can modulate the biological response and clinical success of the scaffold [15,25,85,134,83,132,[121], [123], [195]]. During the past decade, several strategies for scaffold fabrication have been reported (Fig. 4). They include hydrogel-based scaffolds [13,101,[196], [197], [198]], fibrous scaffolds [15,68,108,199,200], microsphere, 3D porous or spongy scaffolds [120,129,134,119,94,93].

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Fig. 4

3.5. Hydrogel

Hydrogels are among the most studied biomaterials in drug delivery, regenerative medicine, and tissue engineering [82,83,112,201]. Luo et al. developed a 3D porous hydrogel-based scaffold to heal breast cancer recurrence and fill the cavity to repair the tissue after surgery [121]. The 3D-printed scaffold was fabricated using dopamine-modified alginate and polydopamine (PDA). The in vitro results showed scaffold flexibility, similar to Young's modulus with normal breast tissues, and higher human normal breast cell adhesion and proliferation. In vitro and in vivo data exhibited an appropriate photothermal effect that can inhibit the local recurrence of breast cancer. The regenerative ability of the scaffold was further evaluated in a mouse subcutaneous tumor model. The in vivo results showed the structural stability of the scaffold one month after implantation. Moreover, the photothermal therapy of 3D printed hydrogel had no systemic toxicity to the major organs [121].

The efficacy of hydrogel-based scaffolds has also been reported for hard tissue regeneration. For example, Tateiwa et al. assessed the bone-regenerating potential of a new composite composed of hydroxyapatite (HA), beta-tricalcium phosphate (β-TCP) microsphere/poloxamer 407-based hydrogel, and recombinant human (rh) BMP-2. Their findings demonstrated that this innovative composite outperformed collagen sponge in terms of bone regeneration capacity. This superiority was attributed to the sustained release of rhBMP-2 and the superior quality of BMP-induced bone, as confirmed through both in vitro and in vivo evaluations [99].

Also, due to hydrogels' ability to absorb and release water, the use of polymeric hydrogels provides a moist environment for wound healing while regulating excessive wound exudates [45,149].

Finally, hydrogels derived from decellularized tissue-specific ECM can offer functionalities similar to those of naturally existing ECM. For instance, Dong-Woo Cho et al. demonstrated that ECM-based hydrogels obtained through decellularization hold significant promise for tissue and organ regeneration, including the kidney [218]. Furthermore, in another study, they formulated hydrogels incorporating decellularized ECM sourced from the endometrium-specific layer and the entire uterus, demonstrating their potential as efficient organ-specific biomaterials. Their findings indicated that intrauterine administration of UdECM prompts endometrial regeneration and enhances fertility, highlighting the therapeutic potential of such biomaterials [219].

3.6. Fiber-based scaffolds

Among biomaterial scaffolds, fibrous structures have the most similarity to most tissue organizations by providing similar structures with similar mechanical properties [1,21,23,171,[220], [221], [222], [223]]. Wu et al. evaluated the regenerative ability of a woven fibrous scaffold containing nanofiber PCL yarns and PLA multifilaments for tendon regeneration [85]. The woven scaffold showed appropriate topographical factors, including large pore size and aligned microstructures that significantly improved the mechanical properties of the construct compared with the control groups. Upon seeding the scaffold with (HADMSC)/(HT)/(HUVEC) tri-culture with dynamic conditioning, the results showed the highest upregulation of collagen secretion and tenogenic differentiation [85]. Mengsteab et al. reported the efficacy of fibrous braided structures in enhancing the osteointegration and regeneration of ACL tissue in a rabbit model [88]. Based on the results, using non-degradable polyethylene terephthalate (PET) fibers in the PLLA bioengineered ACL matrix improved strength retention after surgery. They also demonstrated that the application of BMP-2, FGF-2, and FGF-8 combined with fibrous scaffold enhanced regeneration and suppressed inflammation in the knee joint [88].