Open access peer-reviewed chapter - ONLINE FIRST

Collagen Based 3D Printed Scaffolds for Tissue Engineering

Written By

Sougata Ghosh, Bishwarup Sarkar, Ratnakar Mishra, Nanasaheb Thorat and Sirikanjana Thongmee

Submitted: February 22nd, 2022 Reviewed: February 24th, 2022 Published: March 16th, 2022

DOI: 10.5772/intechopen.103914

IntechOpen
Collagen Biomaterial Edited by Nirmal Mazumder

From the Edited Volume

Collagen Biomaterial [Working Title]

Dr. Nirmal Mazumder and Dr. Sanjiban Chakrabarty

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Abstract

Tissue grafting is mostly used for repair and replacement of severely damaged tissues, the key challenges are compatibility, availability of the grafts, complex surgical process and post-operative complications. Hence, additive technologies such as three-dimensional (3D) bioprinting have emerged as promising alternative for tissue engineering in order to ensure safety, compatibility, and rapid healing. The aim of this chapter is to give an elaborate account of 3D printed scaffolds for bone, cartilage, cardio-vascular and nerve tissue engineering. Various components such as polycaprolactone, poly (lactic-co-glycolic acid), and β-tricalcium phosphate, bioglass 45S5, and nano-hydroxyapatite are combined with collagen and its derivatives to achieve specific pore size in the scaffolds for effective restoration of the defects of soft or hard tissues. Likewise, proanthocyanidin, oxidized hyaluronic acid, methacrylated gelatin, are used in collagen based 3D printed scaffolds for cartilage tissue engineering. Bioink with collagen as active component is also used for developing cardio-vascular implants with recellularizing properties. Collagen in combination with silk fibroin, chitosan, heparin sulphate and others are ideal for fabrication of elastic nerve guidance conduits. In view of the background, collagen-supplemented hydrogels can revolutionize future biomedical approaches for the development of complex scaffolds for tissue engineering.

Keywords

  • biomaterial
  • collagen
  • scaffolds
  • 3D printing
  • tissue engineering
  • regenerative medicine

1. Introduction

Biomedical application of nanotechnology has revolutionized tissue engineering as it can generate efficient biocompatible scaffolds with tuneable physico-chemical properties. Controllable biodegradability is one of the most important aspects as it supports the cells to produce extracellular matrix and promote effective healing. Likewise, adjustable pore structures of the scaffolds provides attractive site for loading drugs for resisting post-surgical infections and promoting cell attachment and colonization. Excellent biomechanical properties obtained by rational selection of the bioink help to mimic the tissue microenvironment and provide load bearing capacity to the tissue after repair [1]. Adherence of cells, proliferation and induction of osteogenic differentiation is higher when the total porosity of the 3D printed surfaces are more than 90% [2, 3]. Hence, such scaffolds with architectural specificity to the desired tissue like bone, cartilage, heart or nerves is immensely critical during implantation in order to ensure regeneration of the new tissue followed by repair [4].

Complete healing in traumatic injury is often a challenge that requires complicated surgical procedures which are often associated with failures and post-surgical infections. Till date bone grafting using autografts, allografts, xenografts, and synthetic bone grafts are employed for fixing the injury [5, 6]. However, the factors critical for success of grafting are the optimal size, shape, biomaterial and the anatomical structure of the bone defects. Thus, 3D printed scaffolds or synthetic bone grafts are considered more feasible due to their tuneable mechanical properties identical to the original bone tissue, and ease of rapid re-vascularization [7].

Weakening and gradual damage to cartilage may also lead to joint injury. Likewise, sudden traumatic injury, formation of lesions and developmental defects may also result is degradation of cartilage and impairment of its function [8]. In the United States alone, it is estimated that around 200,000–300,000 patients have undergone cartilage surgery [9]. It is important to note that the articular cartilage is non-neural, lymphatic, and avascular, having very low self-regenerating capacity [10]. Hence 3D printing mediated fabrication of scaffolds for repair or replacement is thought to be one of the most preferable technologies for cartilage tissue engineering [11]. Similarly, in treating cardiac dysfunctions, it is essential to maintain and mimic the cardiovascular anatomy while fixing the heart defects using tissue engineered vascular grafts (TEVGs). The 3D printing has tremendously helped to fabricate patient- and operation-specific vascular grafts [12]. Further, growing cases of neurodegenerative diseases also require effective therapeutic interventions, which are ideal for axonal regeneration and functional recovery for brain and spinal cord injury (SCI). Neuroregenerative scaffolds developed by 3D printing are considered as innovative materials that mainly focus on providing supportive substrates to guide axons and break the physical and chemical barriers, thereby promoting healing [13].

Collagen type 1 is most favorable for microextrusion based 3D bioprinting of biodegradable and biosorbable scaffolds. Collagen type 1 is the most predominant protein in the extracellular and intercellular matrix, constituting 20–30% of the vertebrate connective tissue, alongside hyaluronic acid (HA). Most importantly, the biocompatibility and low antigenicity of the collagen is attributed to the repeating motifs formed by the alpha chain of hydroxyproline-proline-glycine [14]. Collagen provides highly porous structure and hence permeability which in turn facilitates adhesion, migration, differentiation in addition to the regulation of the cellular morphology [15, 16].

This chapter highlights the collagen based 3D printed scaffolds with their attractive properties such as hydrophilicity, biodegradability, permeability, plasticity and biocompatibility critical for tissue engineering.

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2. Collagen based 3D bioprinting of tissues

Biomaterials composed of collagen as listed in Table 1 are considered ideal substrate for 3D printing mediated fabrication of scaffolds for tissue engineering purposes [32]. However, simulation of the tissue microenvironment is crucial to mimic the physical and morphological properties of the native tissues in order to ensure proper restoration and replacement. The following section elaborates various advances of 3D bioprinting with collagen for tissue engineering.

TissueBiomaterialsReference
Bonepolycaprolactone (PCL), poly (lactic-co-glycolic acid) (PLGA), and β-tricalcium phosphate (β-TCP), atelocollagencircular calvarial defects in male Sprague–Dawley rats[17]
Bonecalcium phosphate, Phosphoric acid, collagencritically sized murine femoral defect[18]
BoneBioglass 45S5 (BG), methacrylated collagen (CMA)human mesenchymal stem cells[19]
Bonemesoporous bioactive glass (BG) microspheres with 4% molar percentage of strontium, Type I collagensimulated body fluid (SBF)[20]
Bonerod-like nano-hydroxyapatite particles embedded in a type I collagen matrix[21]
Cartilagecollagen, oligomeric proanthocyanidin, oxidized hyaluronic acidrat bone marrow mesenchymal stem cells (rBMSCs), bone defects in skulls of the Sprague Dawley (SD) rat[22]
Cartilagemethacrylated gelatin (GelMA), nanohydroxyapatite (nHA)bone marrow mesenchymal stem cells (BMSCs), rabbit osteochondral defect[23]
Cartilagecrude collagen extracted from tendons of skeletally mature rat tailsprimary meniscal fibrochondrocytes[24]
Heart valvegelatin support gel 3D printed with Lifeink® 200subcutaneous implantation in Sprague–Dawley rats[25]
Neonatal scale
human heart
gelated collagenhuman stem cell–
derived cardiomyocytes
[12]
Cardiac tissuegelatin, gum arabic microparticles, rat collagen-Ihuman induced pluripotent stem cells (hiPSC)-cardiomyocytes[26]
Nervecollagen, silk fibroinneural stem cells (NSCs), spinal cord injury (SCI) in Sprague–Dawley rats[27]
Peripheral nervepoly-lactic acid (PLA), collagenPC-12 cells, Schwann cells, and primary chick dorsal root ganglia[28]
Elastic nerve guidance conduits (NGCs)poly(lactide-co-caprolactone) (PLCL), collagen hydrogelsciatic nerve injury models in rats[29]
Neural tissueVEGF-releasing fibrin gel, Type I collagenC17.2 cells[30]
Axonchitosan, collagenspinal cord injury (SCI) in rats[31]
Spinal cordheparin sulfate, collagenneural
stem cells (NSCs) from embryonic day 14 (E14) brains, spinal cord injury (SCI) in rats
[13]

Table 1.

Collagen based biomaterials for 3D printed tissues.

2.1 Bone

Collagen based scaffolds are widely used for bone tissue engineering. Hwang et al. (2017) fabricated bone grafts employing 3D printing using a composite of polycaprolactone (PCL), poly (lactic-co-glycolic acid) (PLGA), and β-tricalcium phosphate (β-TCP) mixed in a ratio of 4:4:2 [17]. Figure 1 shows the scanning electron microscope (SEM) images of the bone grafts. The bone graft developed by solid freeform fabrication (SFF) technique were further mixed with 3% atelocollagen and poured into a mold and incubated at 37°C for 15 min followed by deep freezing for 6 h and freeze drying for 12 h. The collagen based biomaterial was then immersed in ethanol/water (90% v/v) co-solvent containing 50 mM of 1-ethyl-3-(3-dimethyaminopropyl) carbodiimide (EDC) and 20 mM of N-hydroxysuccinimide (NHS) for 24 h at room temperature for effective cross-linking. Each cross-linked collagen block had a diameter and height of 8 mm and 2 mm, respectively. Circular calvarial defects of 8 mm diameter were created by removal of periosteum in male Sprague–Dawley rats. The PCL/PLGA/β-TCP composite block bone grafts were implanted into the defect cites. Interestingly the bone grafts were surrounded by fibrous connective tissues. Subtle bone formation was noted while infiltration of the giant cell and inflammatory cells were seen. However, after eight weeks both neovascularization and new bone formation were noted around the bone grafts. It was speculated that these novel PCL/PLGA/β-TCP composite block bone grafts may be considered as an alternative to synthetic bone grafts.

Figure 1.

SEM images of PCL/PLGA/β-TCP particulate bone grafts. (a) Well-defined PCL/PLGA/β-TCP particulate bone grafts were confirmed at a magnification of ×100; (b) rough surface of PCL/PLGA/β-TCP particulate hone grafts were observed at a magnification of ×800. Reprinted from Hwang et al. [17].

In another study, Inzana et al. tailored a composite scaffold using calcium phosphate and collagen for bone tissue regeneration [18]. Phosphoric acid at a concentration of 8.75 wt% was used as a binder that significantly improved the cellular viability. Tween 80 supplementation further enhanced the strength of the 3D printed scaffolds. Further, supplementation of the binder solution with 1–2 wt% collagen significantly enhanced the maximum flexural strength and cell viability. The pore size was in range from 20 to 50 μm that may significantly facilitate in-growth of the bone and reestablishment of the marrow compartment. The surface was covered by plate like crystal growth which increased the surface area significantly that is ideal for adsorption of drugs and/or proteins. On implanting the 3D printed scaffolds into a critically sized murine femoral defect for 9 weeks, promising osteoconductive properties were noticed.

Kajave et al. (2021) developed a bioactive ink composed of Bioglass 45S5 (BG) and methacrylated collagen (CMA) for 3D printing of biomimetic constructs for bone tissue engineering [19]. The bioink resembled native bone tissue in the organic and inorganic composition. Superior stability with minimum swelling of the collagen based hydrogel was achieved due to homogeneous dispersion of BG particles within the collagen network. Excellent rheological property was confirmed by the betterment in the yield stress. Similarly, incorporation of the BG resulted in improvement in the percent recovery of 3D printed constructs. Additionally, improved bone bioactivity of 3D printed constructs in stimulated body fluid was advantageous. Osteogenic induction and differentiation by BG incorporated CMA (BG-CMA) constructs was associated with high cell viability and enhanced alkaline phosphatase activity and calcium deposition in human mesenchymal stem cells.

In another interesting study, Montalbano et al. fabricated a hybrid bioactive material suitable for 3D printing of scaffolds mimicking the natural composition and structure of healthy bone [20]. Initially mesoporous bioactive glass (BG) microspheres with 4% molar percentage of strontium were synthesized. Thereafter, Type I collagen and strontium-containing mesoporous BG were combined to obtain suspensions able to perform a sol–gel transition under physiological conditions. The fibrous nanostructures were homogeneously distributed embedding inorganic particles as evident from the field emission scanning electron microscopy (FESEM). Large calcium phosphate deposition was observed while release of strontium ions from the embedded BG was attributed to the high-water content of the composite. These features can cumulatively promote the osteogenic induction which is significant for bone tissue engineering. On soaking the composite scaffolds in simulated body fluid (SBF), hydroxyapatite (HA) crystals were uniformly distributed along the cross section of the sample that increased with time from 3rd to 7th day as evident from Figure 2.

Figure 2.

Cross-sectional FESEM images showing HA crystal deposition on collagen/MBG_Sr4% samples after three and seven days of incubation in SBF at different magnifications. Reprinted from Montalbano et al. [20].

In subsequent study Montalbano et al. reported composite biomimetics comprised of rod-like nano-hydroxyapatite particles embedded in a type I collagen matrix [21]. This composite was developed to mimic the bone composition. Initially a hydrothermal method using 0.2% ammonium-based dispersing agent (Darvan 821-A) was employed for the fabrication of the HA nanorods that were uniform-sized with length of 40–60 nm and a width of 20 nm. On suspending this material in a collagen solution in presence of Darvan 821-A, a uniform collagen/nano-HA suspension was obtained that was ideal for extrusion 3D printing. The mesh-like structures printed in a gelatine-supporting bath led to fabrication of 3D bone-like scaffolds.

2.2 Cartilage

One of the most prevalent tissue damages suffered by adults, children and adolescents is articular cartilage defects. In severe cases degenerative joint diseases may result due to exposure of bone terminals caused by progressive wear and tear of articular cartilage. However, low rate of tissue regeneration and self-repairing capacity poses a challenge for effective healing and restoration of the function. Several collagen based 3D scaffolds are being developed for inducing cartilage regeneration that is discussed in detail in this section. Recently, Lee et al. fabricated a highly biocompatible collagen/oligomeric proanthocyanidin/oxidized hyaluronic acid (C/OPC/OHA) composite scaffold with superior compressive strengths between 0.25–0.55 MPa [22]. The composite scaffolds were 3D printed using four types of needles, 25G red plastic, 22G blue plastic, 25G red metal, and 22G blue metal to achieve 20%, 25%, and 30% porosities when pressure of 25, 15, 125, and 100 kPa were applied, respectively as illustrated in Figure 3. Porous nature of the scaffolds is advantageous for promoting both angiogenesis and cartilage ossification. The minimum and maximum storage moduli of the hydrogel were approximately 2.6 kPa and 4.1 kPa, respectively. Interestingly, an increased degradation rate of the composites was 26.6%, 30%, and 30.7% for 0, 5, and 10 mg/mL of OHA, respectively after 49 days. Higher apatite deposition on the scaffold surface was evident on day 21 on immersion in simulated body fluid. Superior cell viability (up to 90%) was achieved when rat bone marrow mesenchymal stem cells (rBMSCs) were grown on the composite scaffolds. On implantation of the scaffolds into bone defects in skulls of the Sprague Dawley (SD) rat, angiogenesis and new bone formation was evident that indicated 3D collagen-based scaffolds could be used as potential candidates for articular cartilage repair.

Figure 3.

Optimization of 3D bioprinting parameters for obtaining porosity at 20%, 25%, and 30% using different needle densities (25G red plastic and metal, 22G blue plastic and metal) and different pressures (25, 15, 135, and 100 kPa). Reprinted from Lee et al. [22].

Liu et al. developed a tri-layered scaffold employing extrusion-based multi-nozzle 3D printing technology where the bioink was comprised of 15% methacrylated gelatin (GelMA) hydrogel for cartilage on top layer, a combination of 20% GelMA and 3% nanohydroxyapatite (nHA) (20/3% GelMA/nHA) hydrogel for interfacial layer, and a 30/3% GelMA/nHA hydrogel for subchondral bone at bottom layer [23]. The composite was biodegradable with maximum degradation (61.4%) in 14 days. Interconnected microtubule-like structure of each layer with interconnected spherical pores with a size of about 300 μm was observed. The Young’s modulus increased with the increase in GelMA concentration in the scaffold. The scaffolds were biocompatible with the bone marrow mesenchymal stem cells (BMSCs) while they exhibited effecting healing of rabbit osteochondral defect. Higher cartilage-specific extracellular matrix formation and collagen type II were observed on treatment with the tri-layered scaffolds. Further, effective new tissue formation and even integration with the surrounding tissues indicated their promises for repair of damages in subchondral bone by inducing cartilage regeneration.

In an interesting study, Rhee et al. fabricated 3D printing assisted soft tissue implants with high-density collagen hydrogels as illustrated in Figure 4 [24]. External heating and collagen concentrations of 12.5, 15, and 17.5 mg/mL enhanced the shape fidelity. At the highest printable concentration, the modulus of printed gel was ̴ 30 kPa. Cell viability within the tissue constructs was high and no notable decrease was observed even after 10 days of culturing. Higher infiltration of the fibochondrocytes cells throughout the collagen matrix was found by 10 days. Adherence of the cells on the outer surface of the nascent collagen fibers was prominent while very few cells colonized the spaces between the fibers.

Figure 4.

Printing process of sheep meniscus, (a) CT scan of meniscus, (b) print path of meniscus deposition of collagen hydrogel during printing, (c) 3D printed meniscus. (d) Geometry assessment of constructs. (e) Constructs scanned using Cyberware 3D scanner. (f) Geometry of the test construct: Half-cylinder. Reprinted with permission from Rhee et al. [24]. Copyright © 2016 American Chemical Society.

2.3 Cardiac tissue (heart)

Cardio-vascular defects such as aortic valve disease (AVD) require high precision surgical procedure that include either mechanical or bioprosthetic valve replacement. Recently, tissue engineered heart valves (TEHV) have gained more attention that are effectively achieved by 3D bioprinting. Maxson et al. evaluated the recellularization potential of 3D-bioprinted scaffold and investigated its applicability as a heart valve implant [25]. Allogenic rat mesenchymal stem cells (rMSCs) with green fluorescent protein (GFP) label were grown and mixed with Lifeink® 200 to obtain a homogenous bioink. Thereafter, a computer aided design (CAD) model for the implant disk scaffolds was prepared wherein the dimensions of the scaffold facilitated easy implantation and mounting in order to avoid migration and folding. Neovascularization was observed after 4 weeks with integration of host tissues with the bioink explants. Moreover after 8 weeks, minimal difference between the two layers was observed; however, the structural integrity of the extracellular matrix (ECM) was maintained. Furthermore, Mason’s trichome revealed fibrosis on the cutaneous side of the explant whereas CD3 and CD163 biomarkers demonstrated chronic inflammation as well as ECM remodeling whose expressions were decreased with subsequent increase in incubation period. CD163 displayed a steady reduction in expression from week 1 to week 8, respectively. On the other hand, CD31 biomarker expression was considerably increased within the same time period due to endothelialization and angiogenesis. The vimentin (a major intermediate filament of smooth muscle cells) concentration of surrounding tissues was also increased with improvement in elastin concentration. This was attributed to the infiltration of the Bioink by interstitial-like cells. In addition, the ultimate tensile strength (UTS) was decreased from 0.344 ± 0.120 MPa in the second week to 0.169 ± 0.077 MPa in the fourth week while it was increased to 0.275 ± 0.166 MPa in the eighth week. Likewise, the tensile modulus was also reduced from 1.186 ± 0.872 MPa in the second week to 0.548 ± 0.341 MPa in the fourth week followed by an increase to 1.425 ± 0.620 MPa in the eighth week. Elastin concentration was significantly increased in the fourth week. Post eight weeks of implantation, expression of CD31 biomarker continued to decrease while CD163 expression increased in week 12 which was attributed to M2 macrophage infiltration. Additionally, the bioink explant was encapsulated by the fibrotic tissue within week 12 while UTS was further increased within this time period. Enhanced levels of both vimentin and elastin indicated strengthening of the extracellular matrix in the bioprinted scaffold due to active collagen deposition. Hence, collagen-based bioink application was demonstrated to be efficient for formation of heart valves.

In another study, Lee et al. also demonstrated 3D bioprinting of collagen for human heart engineering [12]. Herein, 3D bioprinting was carried out using a second generation of the free form reversible embedding of suspended hydrogels (FRESH v2.0) that provides support for printing and then subsequently melts away at 37°C. Moreover, uniform gelatin microparticles with spherical morphology (with diameter ̴ 25 μm) reduced polydispersity. An optimal balance between the resolution of individual strand and strand-to-strand adhesion was further maintained using a 50 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) buffered bath with pH 7.4 which in turn facilitated multiple bioink printing. A linear small coronary artery-scale tube was then fabricated using collagen type I perfusion system with an inner diameter and wall thickness of 1.4 mm and 300 μm, respectively. Thereafter, C2C12 cells were perfused in the tube that displayed viability along with active remodeling of the gel after five days. Further, cellular infiltration was also analyzed using fabrication of collagen disks with a thickness of 5 mm and a diameter of 10 mm wherein excessive cellular infiltration as well as collagen remodeling was observed post three days of implantation in the printed collagen as compared to solid-cast collagen. Moreover, fibronectin and vascular endothelial growth factor (VEGF) were incorporated into the bioink for enhanced vascularization. An extensive vascular network was observed in the printed collagen disk with red blood cells and CD31-positive vessels having a diameter range of 8–50 μm. Thereafter, collagen bioink was used along with human stem cell-derived cardiomyocytes to FRESH print a left ventricle model wherein around 96% post-printing cell viability was achieved through rapid collagen neutralization. A dense layer of interconnected and striated human embryonic stem cell-cardiomyocytes (hESC-CMs) was obtained after seven days of culturing. A baseline spontaneous ventricle beat rate of around 0.5 Hz was captured that was paced at 1 and 2 Hz using field stimulation. Furthermore, the mechanical integrity of the constructs was demonstrated using a 28 mm tri-leaflet heart valve that was robust enough to withstand air pressure. In addition, a neonatal-scale human heart was also printed using collagen bioink that highlighted the potential of FRESH v2.0 printing technique for fabrication of advanced tissue scaffolds for other organ systems as well.

Collagen-based bio-ink was also demonstrated to be an effective tool for direct 3D printing of human induced pluripotent stem cells (hiPSC)-cardiomyocytes that could then be utilized for cardiac tissue engineering [26]. Cardiomyocytes were differentiated in a 2D monolayer followed by CHIR99021-treatment mediated cell expansion and regular passing. Later on, a rat collagen-I based bioink was used for the encapsulation of cells followed by printing in a support bath composed of complex coacervate gelatin/gum arabic microparticles. The bioink was then gelated at 37°C and cultivated under free-floating conditions for a time period of thirty days. Ring-shaped cardiac tissues were printed with 5 × 5 × 1 mm dimension wherein the initial contractions were seen post three days of culturing. Striated sarcomeres were demonstrated with significant responsiveness toward pharmacological stimulations. Therefore, this study demonstrated potential of cardiac tissue engineering with enhanced properties and functions through 3D-bioprinting.

2.4 Nerve

Scaffolds rationally fabricated employing 3D bioprinting could help in the treatment of spinal cord injury (SCI) by nerve tissue engineering. In a study by Jiang et al., Collagen/silk fibroin scaffold was 3D bioprinted and combined with neural stem cells (NSCs) to promote nerve regeneration [27]. A collagen/silk fibroin ratio of 4:2 was used for scaffold preparation using a 3D-bioprinter with a nozzle diameter of 210 μm, printing speed of 9 mm, extrusion speed of 2-mm/min, 0.1 mm thickness and a platform temperature of −20°C. Characterization of the 3D bioprinted scaffolds in rats revealed complete degradation of the composite scaffold after 4 weeks of implantation. Furthermore, the scaffold had considerable ductility as well as compression resistance with a compressive elastic modulus of 60.05 ± 5.12 kPa. Fourier transform infrared (FTIR) spectroscopy results then revealed presence of absorption peaks at 3445.7, 2932.46, 1640.58, and 1376.45 cm−1 that corresponded with -OH or -NH peak, methyl or C-H stretching vibrations of methylene group, C=O or C=C stretching vibrations, and saturated C-H bending vibration, respectively. Hence, these functional groups suggested presence of suitable lipid- and water-soluble bonds in the 3D bioprinted scaffold that may facilitate adhesion and growth of nerve cells. Moreover, significant biocompatibility between the scaffold and NSCs were attained with evenly distributed micropores and pore connections in the scaffold as observed in scanning electron microscopy (SEM) images. Fusiform-shaped cells grew in the scaffold pores, while some cells grew densely on the scaffold surface with extended pseudopods facilitating cell adhesion, growth as well as provided a carrier and channel for regeneration of the nerve fibers. Hence, a conducive microenvironment for NSC adhesion, growth and differentiation was provided by the 3D-bioprinted scaffold. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay also demonstrated successful seeding and proliferation of the NSCs on the scaffold. Thereafter, behavioral changes at the spinal cord injury site were investigated after implantation of the scaffold. The Basso-Beattie-Bresnahan (BBB) open-field locomotor score of the group implanted with 3D-collagen/silk fibroin scaffolds and NSCs was higher as compared to the control after 8 weeks of surgery. In addition, motor function recovery was better in groups having the scaffold and NSCs. Similarly, electrophysiological studies revealed prominent recovery in groups having 3D bioprinted scaffold along with NSCs as compared to control groups. Left hind limb amplitude was significantly higher in scaffold group when compared with control after 1 month of surgery. In addition, magnetic resonance imaging (MRI) and diffusion tensor imaging revealed improved filling of the injury cavity, enhanced spinal cord continuity, increased regenerative axons as well as reduced glial scarring in groups implanted with the scaffold and NSCs.

In another study, Li and Gao fabricated 3D microtubular collagen scaffolds and investigated its potential in peripheral nerve repair [28]. Melt spinning or 3D printing using poly-lactic acid (PLA) was carried out to obtain fibrous template material with a diameter range of 50–100 μm that was then utilized for fabrication of collagen scaffolds. Microtubules were prepared by parallel stacking of melt spun PLA fibers followed by polymerization of the collagen whereas PLA fibers with a diameter of 200 μm and 100 μm interspacing was fused and deposited using 3D printing. The thickness of inner ranged from 10 to 20 μm while the exterior wall formed a shell with a thickness of about 70 μm. Furthermore, cell adhesion ability of adrenal phaeochromocytoma (PC-12) and D62PT Schwann cells was evaluated wherein the cells firmly attached to native as well as chloroform-exposed Matrigel films. Two crosslinkers namely, 0.3% genipin and 0.3% glutaraldehyde were used that decreased swelling as well as enzymatic degradation of the Matrigel. Untreated gels demonstrated retention of 34.5% of total mass after 24 h incubation with 0.05% collagenase, whereas genipin and glutaraldehyde treated gels showed total mass retention of 96.7% and 99.3%, respectively. PC-12 and D62PT Schwann cells further showed well adherence and confluent growth onto microtubule scaffolds after 10 and 4–5 days of culturing, respectively. Moreover, a strong alignment of cells as well as formation of channels was seen in Schwann cells while primary chick dorsal root ganglia displayed neurite growth along the major axis of the microtubes.

Likewise, Yoo et al. reported fabrication of elastic nerve guidance conduits (NGCs) using poly(lactide-co-caprolactone) (PLCL) along with a 3D printed collagen hydrogel [29]. A dense acidified collagen solution with a viscosity of 1.3 × 105 mPa s was used as the bio-ink to print onto the electrospun PLCL membrane that had an optimal porosity of 2.7 ± 0.6 μm which allowed nutrient and oxygen exchange only. The acidified collagen hydrogel was then neutralized using ammonia vapor which prevented crumbling of the hydrogel. Thereafter, the NGCs were shaped into tubes and implanted in the rat sciatic nerve model. SEM images of the longitudinal cross-section of the NGCs demonstrated consistent gel deposition wherein the pore size was reduced by extraction of nano-sized fibers which in turn, prevented cell penetration into the NGCs. Moreover, a conduit fill ratio of 72 ± 2% was observed based on the hydrated cross-sectional images. Furthermore, the biocompatibility of the prepared composite was evaluated using PC12 cell culturing on the PLCL membrane with the 3D printed collagen hydrogel. After 1 week of PC12 cell culturing, a neuron-like elongated differentiation was observed in cells that were grown on the PLCL membrane having 3D printed collagen hydrogel whereas no such differentiation was observed in cells cultured on native PLCL membrane. In addition, no significant differences in the weight percentage of different animal groups were observed as well as no signs of infection, delayed wound healing, or auto-mutilation was observed throughout the experiment. The ankle contracture angles of 3D printing group after 12 weeks of nerve reconstruction was 89.68 ± 2.37% as compared to 93.52 ± 3.17% and 83.86 ± 4.64% for the autograft and bulk collagen groups, respectively. Likewise, the active ankle angle at terminal stance (ATS) was improved in 3D printing and autograft groups after twelve weeks of nerve reconstruction with angle values of as compared to 24.02 ± 1.26° and 19.65 ± 4.78°, respectively as compared to 11.35 ± 2.91° in the case of bulk group. Hence, it was proved that 3D printed collagen hydrogel facilitated motor regeneration using NGCs. Furthermore, a comparable tetanic force of tibialis anterior (TA) muscles was observed in the 3D printing and autograft groups after twelve weeks while the bulk group displayed a lower tetanic force. The nerve regeneration through NGCs was observed after twelve weeks of surgery with linear guidance of the 3D printed collagen hydrogel from the proximal to distal ends along with an organized pattern of the regenerated axons. Moreover, the myelinated axon counts as well as thickness of myelin in the 3D printing group was higher than the bulk group. Additionally, the myelin fiber area and nerve fiber density of 3D printing group were 53,134 ± 5893 μm2 and 11,206 ± 1980 n mm−2, respectively.

Lee et al. also demonstrated bio-printing of collagen and VEGF-releasing fibrin gel scaffolds and investigated its potential in artificial neural tissue construction [30]. Murine neural stem cells (NSCs) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and further used for cell printing. Type I collagen was then prepared and 1.16 mg/mL of the collagen scaffold was used for 3D bio-printing of C17.2 cell-scaffold complex. An average of 56 ± 9 cells/droplet was obtained with a cell viability of 93.23 ± 3.77% which was similar to that of manually-plated cells. Moreover, a collagen scaffold concentration of 1.74 mg/mL demonstrated highly dense and proliferating cells with a viability of 96.72 ± 3.58% after 3 days of culturing. Furthermore, the combinatorial effect of collagen scaffold and VEGF-containing fibrin gel on C17.2 cells was investigated wherein, the cell morphology altered after two days of culturing with active proliferation and formation of clusters. In addition, the cells located near the fibrin gel border gradually migrated toward the VEGF-containing fibrin gel and continued differentiation. After three days of culturing, the total migration distance was 102.4 ± 76.1 μm. Hence, proper cell proliferation and migration was displayed using the two scaffolds which highlighted the potential of 3D bioprinting in artificial tissue construction.

Likewise, axon regeneration was ameliorated by Sun et al. using 3D printed collagen/chitosan scaffolds [31]. A 3D bioprinter was used for fabrication of the scaffold that had an interconnected porous structure with a porosity of 83.5% as observed in SEM images. The pore size of the scaffold ranged from 60 to 200 μm. Hence, significant space was obtained by the cells for growth and adherence. The compressive modulus of 3D collagen/chitosan scaffold was 3.82 ± 0.25 MPa along with enhanced compressive strength of 345.20 ± 29.60 KPa. The cytocompatibility of 3D printed scaffolds was similar to that of scaffolds prepared using freeze drying technology. Interestingly, the persistent locomotion recovery as well as significant increase in blood brain barrier (BBB) scores was observed after implantation of the 3D printed collagen/chitosan scaffolds in rats with spinal cord injury (SCI). Moreover, the magnetic resonance and diffusion tensor imaging results revealed a significant signal increase at the epicenter of the spinal cord lesion in rats implanted with 3D printed collagen/chitosan scaffold. Post eight weeks of SCI surgery, the axonal regeneration was demonstrated wherein 3D collagen/chitosan implantations resulted in amplitude and latency improvement. Further confirmation of axonal regeneration was carried out using anterograde biotin dextran amine (BDA) labeling wherein BDA-positive fibers were observed in 3D collagen/chitosan implantations. Hematoxylin and eosin (HE) staining also demonstrated linear ordered structure of the spinal cord after eight weeks with no obvious cavity observed in 3D printed collagen/chitosan implanted group whereas visible cavities and disordered structures were observed in injury groups. Hence, 3D printed scaffolds were demonstrated to be effective in axon regeneration and amelioration of spinal cord injury.

In a similar study, Chen et al. constructed collagen/heparin sulfate based scaffolds using 3D bioprinting and evaluated its action in functional SCI recovery in rats [13]. The scaffold was prepared using a 3D bioprinter that had a cylindrical morphology with a uniform and regular internal structure along with high porosity as observed in SEM images. The compressive modulus of 3D printed collagen/heparin sulfate was 3.46 ± 0.278 MPa which was higher as compared to scaffolds prepared using freeze drying technology. Likewise, enhanced compressive strength of 308.9 ± 28.65 KPa was observed in 3D printed scaffold. Furthermore, release profile of basic fibroblast growth factor (bFGF) from 3D printed scaffold was also evaluated wherein scaffolds prepared using freeze drying method demonstrated an initial burst of 54.89% of bFGF was released in the first day after which a slow release behavior was observed for longer time period. However, a steady bFGF release behavior was observed in case of 3D printed scaffolds for twenty days. Thereafter, the biocompatibility of scaffolds was analyzed using NSCs which proliferated inside the pore followed by spreading on the wall of the scaffolds. In addition, MTT assay revealed no significant difference in cell growth on different scaffolds thus highlighting the cytocompatibility of the 3D printed collagen/heparin sulfate scaffolds. Implantation of the 3D printed scaffolds further demonstrated significant recovery of locomotor functions in rats after two months with amelioration of the SCI as well as enhanced number of neurofilament positive cells.

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3. Conclusions and future perspectives

Advances in the field of nanomedicine have enabled exploration of novel biomaterials for tissue engineering. Among various biopolymers such as, chitosan, alginate, silk fibrion, collagen is considered as most attractive due to its biocompatibility and biodegradability. However, high temperature and extreme conditions during fabrication and bioprinting results in low stability of the collagen molecules. Hence, ideal porous scaffolds should involve combination of type I collagen and hydroxyapatite particles by freeze-drying. It is essential to have tuneable pore dimensions for superior ingrowth of cells and blood vessels. More complex microarchitectures of the collagen based scaffolds with specific rheological properties such as shear thinning, yield stress and fast shear recovery can be obtained using extrusion-based 3D printing [33].

Various biologically synthesized nanoparticles like silver, gold, copper, platinum, palladium and others can be supplemented in the scaffolds resisting post-surgical microbial infections [34, 35, 36, 37]. Biofilm associated infections are most challenging to treat and are highly responsible for implant failure. Hence, coating of implants with antimicrobial nanoparticles impregnated collagen can be an effective strategy to increase the shelf life of the implants [38, 39]. Also drug functionalized nanoparticles can be embedded in the collagen matrix to ensure sustained release and rapid healing of the injured tissues.

Multiple approaches and integration of medical biology and material science will certainly help to revolutionize regenerative medicine by rational tissue engineering. In view of the background collagen based 3D printed scaffolds hold tremendous potential as candidate nanotherapeutics.

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Acknowledgments

Dr. Sougata Ghosh acknowledges Kasetsart University, Bangkok, Thailand for Post Doctoral Fellowship and funding under Reinventing University Program (Ref. No. 6501.0207/10870 dated 9th November, 2021).

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Conflict of interest

The authors declare no conflict of interest.

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Written By

Sougata Ghosh, Bishwarup Sarkar, Ratnakar Mishra, Nanasaheb Thorat and Sirikanjana Thongmee

Submitted: February 22nd, 2022 Reviewed: February 24th, 2022 Published: March 16th, 2022