Three-Dimensional Printing Technology Combined with Materials

Chapter 13

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Three-Dimensional Printing Technology Combined with Materials Drives Meniscal and Cartilaginous Regeneration Zhu-Xing Zhou, Zheng-Zheng Zhang, Shao-Jie Wang, Dong Jiang, and Jia-Kuo Yu* Institute of Sports Medicine of Peking University, Peking University Third Hospital, No. 49 North Garden Road, Haidian, Beijing, China 100191 *E-mail: [email protected].

Meniscal and cartilaginous injuries are both common diseases of knee joints, which tend to be great risk factors for the degenerative joints or osteoarthritis (OA). Osteoarthritis, which is the most common type of arthritis, is a disabling disease affecting many adults and old ages and weakening the quality of life of its victim. Unfortunately, there is no cure for OA. Thus, irreversible meniscal or cartilaginous disorders are in urgent need of novel and appropriate treatments to avoid the development of OA. Regenerative medicine is offering the new therapeutic strategies for irreversible meniscal or cartilaginous disorders, aiming at restoring or replacing the destroyed cells or tissues by tissue engineering technique. Recently, with the rapid development of three-dimensional (3D) printing technique in the field of regenerative medicine, it is possible to regenerate meniscus and articular cartilage by combining 3D printing technique with tissue engineering. Despite of some challenge, the developing 3D printing technology is now coordinating with materials science to bring new prospects for regenerating complete meniscus and cartilage.

Background Injuries to articular cartilages in knee joints caused by trauma is quite common, which may frequently lead to the focal chondral defects. It was reported © 2017 American Chemical Society Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


that an increasing frequency of chondral injuries among professional athletes or amateur athletes or just sports enthusiasts were observed in high-level competitive sports or organized recreational sports activities (1, 2). Articular lesions or defects will interfere those athletes’ training schedules or daily lives, due to the subsequent unconformable symptoms, such as pain, locking, catching, and swelling (2). Articular cartilage with its unique structures lacking vascularization and containing a few dispersed chondrocytes has the intrinsic limited potential of healing spontaneously after injury (3). Some of the articular cartilage lesions are associated with meniscal injuries. Menisci, semi-lunar fibrocartilaginous tissues with complex microstructures between the tibial plateau and femoral condyles in knee joints, maintain the stabilization and homeostasis of the knee and also protect articular cartilage by preventing focal cartilage from the concentration of stresses (4). Traumatic meniscal disorders are also very common. In the United States, meniscal tear is one of the most frequently sports medicine injuries (5). It is very difficult for the inner region of meniscus without vascular distribution to heal after tearing. To relieve the symptoms caused by meniscal injuries, partial or total meniscectomy is commonly used to deal with irreparable meniscal tears (6). However, the surgery is followed by the onset of degenerative changes in knee joints, because it may increase the stress on the articular cartilages, destructing cartilaginous structures, which most likely contributes to disabilities of knee joints finally (7). Overall, both of the injuries to cartilages and menisci may develop into osteoarthritis progressively, which is often called the post-traumatic osteoarthritis (OA) (8). In contrast, primary OA is often defined as a degenerative disease with elusive pathogenesis, which is the most common type of arthritis affecting many old ages (9). OA, a disabling disease, often causes deformity of joints decreasing the quality of life and leads to considerable health-care expenditures (10). For both primary OA and post-traumatic OA, the feature of the disease is mainly characterized as progressive, irreversible pathological process of cartilage (11, 12). Unfortunately, we have no to date curable treatments, so that many patients with ultimate stage OA have to undergo arthroplasty (13). As mentioned above, patients suffering from either cartilaginous defects or meniscal tears are likely to develop the OA prematurely with high risk, which is one of the main causes of serious physical disabilities in adults, the management of the cartilaginous and meniscal disorders therefore become the focuses of many physicians.

Present Treatment Option for Cartilaginous or Meniscal Injuries Current interventions to treat cartilaginous injuries include two main types, non-surgical or surgical approaches. For alleviating the symptoms of patients with cartilaginous injuries, some conservative treatments can be applied firstly, including nonsteroidal anti-inflammatory medications (NSAIDs), glucosamine and chondroitin and physical therapy (1, 14). 254 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


However, surgical intervention may be necessary if those non-surgical treatments fail to achieve the goal of symptomatic relief (2, 15). Traditionally, surgical techniques are striving to fill the defects of cartilages by lavage, debridement, drilling, or microfracture, aiming at stimulating bone marrow to induce the formation of neo-cartilaginous tissues (16). This technique, which debrides the calcified cartilage layer and penetrates subchondral bone, promoting the mesenchymal stem cells to migrate from bone marrow to the site of cartilage defects. However, the fibrocartilaginous tissues induced by microfractures often prove to be inferior to hyaline cartilages biomechanically and biochemically. Besides, osteochondral allografts or autografts have been devised and investigated to replace rather than repair cartilage defects. For the osteochondral allografts, despite the good outcomes in 75% to 86% of patients, the widespread use of this technique has been limited substantially because of the potential risk of transmitted diseases and difficulties with obtaining fresh, unirradiated grafts (2, 16). Osteochondral autograft transplantation technique has also been used more recently. Some prospective studies reported that 95% good or excellent results, with significantly improved knee function scores after 26–36 months in athletes (15). Autografts are often harvested from a site of the distal femur that experiences the lowest contact pressures, and the defect of the donor site can be restored by bone graft substitute if desired, but donor-site morbidity can be significant. It is noteworthy that peripheral chondrocyte death induced by trauma and the recipient edges may contribute to unsuccessful peripheral integration with the persistent gap formation (2). With the respect to the treatment of meniscal lesions, it has evolved tremendously in the recent years. A huge number of patients with traumatic or degenerative meniscal disorders can be treated non-operatively, however, surgical procedures and repair techniques are still significant challenges for the surgeon, especially for the young active patient with meniscal tear (6). Just as cartilaginous repair, some techniques such as debridement and trephination have been also used to treat meniscal injury. However, their clinical efficacy have not guaranteed exactly (6, 17). More importantly, the possibility of repairing the meniscal tear is supposed to be considered carefully before the operation (7). Currently, it is widely supported that longitudinal tears of the vascular zone must be repaired, because of their relatively better prognosis in most cases (18). However, in the area of avascularity, meniscal repair is intermediate, and it is not uncertain whether the mechanical function of the meniscus will be preserved, thus, meniscal repair in this area is still a challenge (18). Overall, for symptomatic meniscal injuries in stable knees, when the tear is in a zone where surgical techniques fail to repair it, partial meniscectomy is needed, although meniscal tissues are strongly recommended to be preserved in case of the accelerating development of degeneration in knee joints (19). Meniscal transplantation is another alternative approach to relieving the patients’ symptoms with previous complete meniscectomy. The main purposes of a meniscal replacement are to decrease the painful experience of patients following meniscus resection, prevent the knee joints from the degenerative changes, and 255 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


to reduce or avoid the risk of OA (7, 19). There are two types of meniscal allografts for meniscal transplantation: fresh and frozen. Like other allografts, immune reactions and greater risks of infectious diseases are problematic for the widespread use of fresh meniscal allografts (7, 19). In comparison to the fresh transplants, frozen allografts are used most frequently, although it doesn’t prove to protect the hyaline cartilage, or reproduce the meniscal functions (7). After analyzing more than 40 published clinical trials, a recent meta-analysis concluded that meniscal transplantation allowed patients to resume high levels of activity and work with safety and effectiveness (20). Actually, a number of clinical and radiological mid-term results have been evaluated and reported by some researchers (20, 21). However, the procurement of meniscal allografts, the technically demanding surgical procedure, and the frequent mismatch of graft and host tissue limit the use of this technique (22). In conclusion, meniscal transplantation allows for acceptable short-to-intermediate term results in selected patients, but it is not yet demonstrated whether this technique provides long term protection from joint degeneration (23). In other words, no present strategies demonstrated regeneration of a functional, long-lasting meniscal tissues and re-establishment of a proper knee homeostasis in the meniscectomised knee joint (6). In recent years, with the rise of the concept of regenerative medicine (RM), it brings new hope to the regeneration of cartilage and meniscus. Regenerative medicine refers to the treatment that harnesses the human body’s inherent ability to regenerate a tissue at the level of cellular or organ structure that foster cellular communication, translation, organ system refurbishment, and result in overall organism well-being (24). Briefly, the aim of regenerative medicine is to replace or regenerate the human cells, tissues or organs to restore or establish their normal function. At first, a series of cell-based therapeutic strategies have been developed to treat cartilaginous and meniscal lesions. For instance, autologous chondrocytes implantation (ACI) has been proposed to be as a restorative approach for cartilaginous regeneration. To obtain enough chondrocytes, a full-thickness cartilage from a low-weight-bearing region of the joint is harvested through arthroscopic operation and then expanded in vitro, yielding near 50 million cells. Surgeons then will implant these chondrocytes into the debrided cartilage defect and covered by a membrane. One of the major benefits of ACI is that it avoids the possible risk of immune complications and infections leading to transmitted diseases because of those transplanted cells from patients’ own tissues. Another one is that the small lesion of biopsy minimizes injuries and complications for the chondrocyte donor, in contrast to autologous osteochondral implantation. While these strategies have been proved to effectively relieve the symptoms and improve patients’ function, these chondrocytes tend to dedifferentiate or lose their phenotype because of being cultured in vitro, which attenuates their normal function for the regenerative cartilage with inferior biomechanical and biochemical properties (12, 13, 25). There is another attractive strategy for joint regeneration by intra-articular injection of mesenchymal stem cells (MSCs) or progenitor cells directly. In 2008, a group firstly reported that a promising result of the clinical and MRI improvements at early follow-up after single intra-articular injection of autologous bone marrow256 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


derived MSCs (BMSCs) in a patient with knee degenerative cartilage disease (26), and other similar positive results at short term were later shown in the subsequent researches (27, 28). Orozco et al. also concluded that BMSCs injection improved knee OA in the first 12 months effectively (29), and the efficacy was maintained at 24-month follow-up, together with improved cartilage quality at MRI (30). Although those researches have shown a series of encouraging results, the potential benefits of those treatments need to be validated by further clinical data through high-quality studies with longer follow-up. In the studies on meniscal regeneration, Murphy et al. (31) observed that cells succeeded in surviving and engrafting in the regenerated medial meniscus by injecting BMSCs in a goat model established for OA research. In this model, the medial meniscus was complete excised previously. In another animal study conducted by Horie et al., the authors showed positive role of injected synovial membrane-derived MSCs in the process of regenerated meniscus, where stem cells adhered to the local sites and formed neo-tissue (32). These findings are quite intriguing, however, more large animal models are needed to be performed prior to show stronger evidence in order to support this strategy as a potential alternative with clinical relevance. Tissue-engineered technique is regarded as a subfield of regenerative medicine and strictly indicated producing tissues or organs in vitro, by seeding sells on and/or into a supporting substrates (33). Also, tissue-engineered technique is a promising approach with attractive prospects to obtain a complete and fully functional meniscal and cartilaginous tissues. This technique was composed of three main parts, namely living cells, biocompatible materials, and suitable biochemical and biomechanical stimuli (33). The types of seed cells used in the current strategy to regenerate cartilage and meniscus are various, including chondrocytes from nasal, costal, or a non-weight bearing portion of the joint; meniscal cells; bone-marrow derived mesenchymal stem cells, peripheral blood mesenchymal stem cells (PBMSCs), adipose derived mesenchymal stem cells, synovial membrane derived mesenchymal stem cells (34). Our previous study demonstrated that Peripheral blood MSCs were successfully mobilized by the method of combined drug administration, then isolated, expanded, and identified in vitro (35). There is no significant difference between PBMSCs and BMSCs, with respect to the morphology, immune phenotype, and antiapoptotic capacity. In addition, MSCs from both sources compounded with decalcified bone matrix showed the same ability to repair cartilage defects in rabbit model (35). MSCs derived from synovial tissues also have the great potential to regenerate the meniscal tissues. In a recent study on rat model with partial meniscal defect, researchers found that synovial MSCs enhanced regeneration of meniscus, augmented by an autologous achilles tendon graft, and prevented cartilage degeneration as well (36). In order to obtain ideal tissue-engineered products, researchers have gained better understanding of the behaviors of seed cells, like cellular proliferation, differentiation and more importantly, the interaction between cells and the niche during the process of the formation of neo-tissues. extracellular matrix (ECM) contains many bioactive agents, such as proteins, proteoglycans and necessary nutrients for cellular growth (34). It also plays a role of structural support to 257 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


maintain normal cellular functionality such as regulating cellular communication, growth (34). In vitro studies have shown that the well-formed regenerated cartilage is dependent both on the chondrocyte proliferation rate and on the differentiation capacity of stem cells within a tissue-engineered 3D matrix akin to that of ECM (37). Similarly, meniscal cells showed good attachment within the 3D collagen sponge microenvironment and expanded with culture time (38). Thereby, one of the cores of tissue-engineered technique is to intimate the ECM and basically often begins with a scaffold, which is a three-dimensional substrate essential for modulating seed cells to proliferate and differentiate. Biomaterials for fabricating the scaffold have become the research focus of tissue-engineered techniques. The properties of biomaterials are supposed to meet the following requirements with them to be biodegradable, atoxic, biomechanically competent (similar to surrounding tissue), be able to regulate cell activity, which also need appropriate surfaces to facilitate the attachment of cells, and be shaped into different sizes and forms. The types of materials for augmenting the regeneration of cartilage or meniscus are alike: (1) protein-based polymers, (2) carbohydrate-based polymers, (3) synthetic polymers, and (4) composite polymers (39, 40). Although there are abundant materials developed in recent studies on cartilaginous and meniscal regeneration, in which researchers have made some progress, none of them actually can meet all the criteria. Besides, many techniques have been established for scaffold fabrication including solvent-casting particulate-leaching, gas foaming, fibre meshes/fibre bonding, emulsion freeze drying, solution casting, as well as freeze drying. But, these conventional approaches have many intrinsic limitations because they are not adequate at precisely controlling the microstructures of scaffold, including pore size, pore geometry, the levels of interconnectivity, topology mechanical strength (40–42).

3D Printing Technology for Application of Regenerative Medicine In 1986, Charles W. Hull firstly introduced the definition of 3D printing, which also referred to as additive manufacturing (AM) or rapid prototyping (RP). According to the definition given by ASTM (F2792), AM processes include the following parts: binder jetting, directed energy deposition, material extrusion, material jetting, powder bed fusion, sheet lamination and vat photopolymerization (43, 44). By adding materials layer-by-layer, an object with three dimensional can be created, therefore, 3D printing is a vivid description for the technology, and the materials used for fabrication include ceramic, metal, plastic, and polymers (44, 45). The general principles of 3D printing could be summarized as 3 steps, including modeling, printing, finishing. Before printing, the design is supposed to be accomplished with the aid of computer, namely computer-aid design (CAD) 258 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


via 3D scanner (46). One of the major benefits of 3D printing is able to directly transform an idea into an end product in a convenient, economical way (47). 3D scanner will provide the digital data on the shape and appearance of a real object, creating a digital model based on 3D scan. Next, 3D printing apparatus reads out data from the CAD file and then a product with 3D structure is produced. 3D digital models are created with the aid of CAD, therefore, some errors can be corrected and modified before printing to allow repeated confirmation by designer. Stereolithography (SLA), multijet modeling (MJM), selective laser sintering (SLS), and fused deposition modeling (FDM) are the main techniques that have been explored extensively (41, 44). SLA is the earliest 3D printing technology that is described for the fabrication of biomodels, in which a layer of liquid photopolymer in a vat is cured by a low-power ultraviolet (UV) laser (41, 44). SLA has been regarded as the gold standard in 3D biomodel production and can yield resolutions of up to 0.025 mm (41, 44). MJM is similar to SLA, however, the liquid photopolymer is immediately cured by the UV light preventing the time-consuming (41, 44). MJM is able to fabricate products with high resolution increasing the advantages of the products with multiple materials deposited for the desired degree of tensile strength and durability (41, 44). Compared with the SLA equipment, MJM printer is more likely to be maintained (41, 44). But it is more suitable for producing in large scale because of its high cost. If high mechanical strength and low porosity are needed for fabricating products, SLS can be a favorable technique. FDM, one of the most commonly used 3D printing technology for commercial purpose, heats up the thermoresponsive polymers beyond their glass transition temperatures and then polymers will deposit onto a solid medium to build the products. One of the advantages of FDM technique is diminishing the potential toxicity of the organic solvents for solubilizing some polymers (41). However, a thermoplastic material required for FDM limits the application of technology in medical field (41). As a kind of 3D printing, FDM is a cost-efficient rapid prototyping technique in contrast with other 3D techniques like SLA and SLS. Moreover, it is possible for FDM techniques to manufacture a 3D scaffold with its microstructural parameters, such as pore size, porosity, and pore interconnection controlled, which is another advantage that other traditional 3D techniques, like particle/salt leaching and electrospinning cannot match (41). In addition to be less expensive, FDM is a relatively fast, and a widely well-explored technology which can generate 3D scaffolds that are suitable for musculoskeletal disorders, however, due to lacking more new materials developed for FDM use, the application of this technique in tissue engineering is greatly restricted. Currently, Many researchers have explored to reconstruct the complex tissues through 3D printing techniques, like microvasculature printing, muscle printing, bone printing, skin printing, cardiac tissues printing, liver tissues printing, and 3D printing has become a very useful tool in making biomaterial scaffolds with custom-designed geometries, which promotes the great development of tissue engineering technique and regenerative medicine (41, 48, 49). In particular, tissue engineering augmented by 3D printing has a good prospect in addressing the present challenging scenarios of cartilaginous and meniscal loss and may provide a viable alternative to current treatment modalities. 259 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


3D Materials Promote Meniscal and Cartilaginous Regeneration As mentioned above, the repair and regeneration of meniscus and cartilage tissues is one of the most challenging and intractable issues for orthopedic surgeons, which drives clinicians to look for another creative approach to cope with it. The emerging of 3D printing technology has brought intelligent and very useful tools to both doctors and researchers when dealing with such challenging issues of regenerated meniscus and cartilage from bench to bed. In recent years, an increasing number of tissue-engineered products directed by 3D printing technique for meniscal and cartilaginous regeneration have been evaluated and revealed positive results, which is a good sign for generating the functional substitutes for repairing or replacing the injured and even destroyed menisci or cartilages. Lee and his co-workers published an article with a poly (e-caprolactone) (PCL) meniscal scaffold fabricated by 3D-printer and tethered with growth factors: connective tissue growth factor (CTGF) and transforming growth factor–β3 (TGFβ3) (50). In their work, they not only obtained 3D-printed and anatomically shaped meniscal scaffolds, but encapsulated CTGF in the scaffold’s outer/middle zones and TGFβ3 in inner/middle zones. This design idea with spatiotemporal CTGF and TGFβ3 release was to direct the formation of inhomogeneous meniscal tissues by inducing the same endogenous stem cells or progenitor cells to differentiate into cartilaginous tissues in the inner/middle zone, and fibrocartilaginous tissues in the middle/outer zone, respectively. The promising results showed that it was possible to regenerative bionic meniscus with regionally inhomogeneous tissue properties and functions. Based on the interesting results, considering the intrinsically heterogeneous characters of menisci, it is essential to fabricate a scaffold in according with the inhomogeneity of meniscal tissues in order to achieve a well-functioned implant. Besides, a well-designed acellular scaffold with its high-precision structures to be encapsulated controlled-release growth factors is providing a viable alternative and good outlook for regenerated tissues. In addition to the meniscal regeneration, PCL scaffold is also widely used for tissue-engineered cartilage. In a recent study on tissue-engineered cartilage, PCL scaffolds were produced by the 3D printing technique (51). After cross-linking with 3D PCL scaffold, the chondrocytes-encapsulated “soft scaffold” succeeded in anchoring into the PCL backbone to form a PCL-hydrogel-chondrocytes composite scaffold. It has been observed that the formation and synthesis of cartilaginous matrix augmented by the scaffold in this study. Another research adopted electrospinning techniques to fabricate the PCL scaffold construction with chondrocytes suspended in a fibrin-collagen hydrogel (52). In this construct, there were 5 layers of material deposited into a whole with 1mm-thick. Both of the results in vitro and in vivo showed that the formation of cartilage-like tissues, and the synthesis of Col-II and glycosaminoglycan (GAG), which indicated that the feasibility of a complex 3D cartilaginous constructed by a hybrid inkjet printing/electrospinning system. In our recent work, we are also striving for the improvement of regenerative strategy for meniscal and cartilaginous disorders. Next, we would like to 260 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

summarize our recent studies on the combination of 3D printing technology and materials to construct tissue-engineered meniscus and cartilage.


Role of Scaffold Mean Pore Size in Meniscus Regeneration A series of three-dimensional PCL scaffolds with three different mean pore sizes (i.e., 215, 320, and 515μm) were fabricated by FDM (53), as shown in Figure 1. In vitro, bone marrow derived stem cells were harvest from New Zealand Rabbits and then seeded into those scaffolds with 3 different mean pore sizes. The results showed that the different effects of the scaffolds with 3 distinct mean pore sizes on cellular proliferation and the formation of extracellular matrix. Compared with the other two groups (320, and 515μm), the scaffold with the mean pore size of 215μm significantly improved the proliferation of mesenchymal stem cells.

Figure 1. Schematic illustration for evaluation of tissue-engineered meniscus augmented by PCL scaffolds with different mean pore sizes in vitro and in vivo. Reproduced with permission from reference (53). Copyright 2016 Acta Materialia Inc.

In the analysis of the ECM and type II collagen (Col-II) production of stem cells revealed that cells in the 215μm group deposited more ECM and Col-II than the other 2 groups as shown in Figure 2. However, type I collagen (Col-I) was rarely synthesized in all three groups. Biomechanical analysis showed that the scaffolds with mean pore size of 215μm exhibited the greatest tensile and compressive moduli in all the acellular and cellular studies.

261 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 2. Representative 3D micro images of MSCs colonization and Col II deposition in scaffolds with various mean pore sizes (A–C). The surface area covered by live MSCs was the greatest on the 215μm scaffold in all three groups. Scale bar represented 300μm. MSCs colonized and bridged neighboring fibers in the former group, while those placed on the latter were isolated (D–F). Scale bar represented 50μm. The largest areas of synthesized matrices around the pores were shown in the 215μm scaffold compared with the other two scaffolds (G–I). Scale bar represented 300μm. Reproduced with permission from reference (53). Copyright 2016 Acta Materialia Inc. In vivo, acellular PCL scaffolds as meniscus grafts with 3 distinct mean pore sizes were implanted into the right knees of the rabbits to replace the medial meniscus. Twelve weeks after surgery, the rabbits were sacrificed and their knee joints were excised and evaluated. It has been shown that the meniscus was intact in the control knee joints with no sign of degradation, while various degrees of cartilage damage were observed in the operated joints. The operated joints implanted with 215 μm meniscal scaffolds exhibited significantly lower joint degeneration, compared with those of the other groups, as shown in Figure 3. The results of the gross evaluation of meniscal scaffolds suggested that meniscal-like tissues formed in all of the implanted scaffolds and the surface of 215μm scaffolds seemed smoother and to integrate to the joint with no sign of disruption or gap information. In contrast, scaffolds in the other two groups were totally collapsed or were fully deformed, and some scaffolds have not even been identified. In addition, we also carried out histological and immunohistochemical analysis of those scaffold specimens and compared the results. We found that in the 215μm scaffold group, the implant was covered by peripheral synovial cells incorporating into the pores. We also detected that Col-I and Col-II depositions in the 215μm scaffold group were significantly higher than that in the other groups, indicating that the pore sizes of scaffold were probably effective in mediating 262 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


the differentiation of endogenous stem cells or progenitor cells. In terms of the evaluative results of articular cartilage, better results were also obtained in the 215μm scaffold group. Scaffolds in the 215μm group reflected superior chondroprotective effects in both femur and tibia side, as shown in Figure 3.

Figure 3. Macroscopic observations of joints and implants at postoperative 12 weeks. Scale bar represented 10 mm. Reproduced with permission from reference (53). Copyright 2016 Acta Materialia Inc. This study investigated the role of mean pore size of scaffolds in the meniscal regeneration, which provides a unique insight into the effects of pore structures on cellular behaviors and subsequent biomechanical properties, and chondroprotective effects. Based on the research, we found that the pore size of scaffold is an important factor, which not only influenced the mechanical strength as described before, but, more importantly, might direct the fate of stem cells in vitro or in vivo. Therefore, the results highlight the importance of scaffold architectures in the field of meniscal tissue engineering. Given the better proliferative effects of the smallest pore sizes, we speculated that the smallest pore size offered the largest area for cell colonization and cellular proliferation, contributing to the potential downstream effect, such as differentiation and deposition of extracellular matrix. Notably, in addition to the proliferative effect, the fibrochondrogenic differentiation and the deposition of extracellular matrix of mesenchymal stem cells were also influenced by the 263 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


mean pore sizes of meniscal scaffolds in vitro and in vivo. In vitro, the results showed that the neo-tissues formed in the cell-seeded scaffolds were likely to be closer to hyaline cartilages historically with them depositing more Col II and GAG production. Interestingly, fibrocartilaginous tissues were found in the cell-free implants at 12 weeks after implantation in vivo, which was most obvious in the 215μm group. In fact, there is to date a controversy about how to induce the mesenchymal stem cells into the fibrochondrocytes and it was reported that inducing conditions in vitro had inferior effect on fibrocartilaginous formation compared to the in vivo results. It was likely that 215μm pore size of scaffolds with the greatest surface area were beneficial for 3D cell colonization and the bridge neighboring fibers, leading to superior potential in differentiation. Given the potential effects of pore size on the differentiation of stem cells, the microstructures of a scaffold are supposed to be focused because they may be necessary to succeed in constructing the regenerated meniscal tissues close to the native meniscus (50). However, the exact mechanisms of mean pore size affecting cell behavior remain to be unknown that is needed to be explored in further research. Moreover, the biomechanical property is another factor which is supposed to be taken into consideration, because the pore size, porosity, and the interconnection of a porous scaffold couple with the biomechanics. The mechanical properties of acellular and cellular scaffolds with mean pore size of 215μm were both better in tensile and compressive moduli than that in the other two groups. As predicated, the best protection for articular cartilage was provided by the scaffolds with the smallest pore size, because of their strongest biomechanics to resist the stress or load in vivo. It is inevitable for the mechanical properties of these scaffolds to decrease due to the degradation of polymers upon being implanted into the knees. However, the biomechanics of scaffolds will be compensated for the ingrowth of endogenous cells depositing ECM simultaneously. Thus, the mechanical strength of implants not only depend on the initial conditions of scaffolds, but the ingrowth and integration of the native issues, which is linked closely with the pore size. In the past, many traditional techniques could not provide such alteration of conditions, thanks to the great development of 3D printing, precisely controlled microstructures are available during the fabrication of scaffold. Thus, such an idea to explore the response of stem cells/endogenous progenitor cells to the microstructures can be performed by combining the 3D printing technology with materials.

3D Printing PCL Scaffold Augmented with MSCs for Total Meniscal Substitution

In another work, we also made the PCL meniscal scaffolds by FDM technique. The 3D models of the medial menisci of rabbits were established by the Mimics software system, version 17.0 based on the data of magnetic resonance image (MRI) scan, as shown in Figure 4. 264 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 4. (A) Anatomic reconstruction model of rabbit menisci in left knee. (B) A typical model of 3D medial meniscal scaffold. (C) 3D printing PCL scaffold seeded with MSCs (Scale bar represented10 mm). (D) PCL scaffold (lower arrow) implanted between femur and tibia with medial collateral ligament (upper arrow) reserved. In this controlled laboratory study, we mainly observed that the role of the PCL scaffolds with or without stem cells for meniscal regeneration in rabbits. It has been shown that the scaffold loaded with cells revealed significant better gross appearance. Fibrochondrocytes surrounded by extracellular collagen type I, II, III and proteoglycans were detected in the implants of cell seeded and cell free groups, while the results were significant better for the cell seeded group at 24weeks. With regard to the cartilage degeneration, implants in the cell seeded group presented lower cartilage lesions significantly, compared with acellular or meniscectomy group, as shown in Figure 5 and Figure 6. Herein, we demonstrated that bone marrow derived mesenchymal stem cells combined with 3D printing PCL meniscal scaffolds have the potential to regenerate meniscus-like tissues. Mesenchymal stem cells are common sources of seed cells in the field of tissue engineering technique that have been evaluated in many researches. In the research, scaffolds with seeded cells presented better functional results than acellular ones for meniscal regeneration, which suggested that cell-based scaffolds could be more beneficial for tissue-engineered menisci than that of cell-free ones. It was observed that better fibrocartilaginous formation and lower foreign-body reaction were shown in the group of scaffolds with seeded cells in our study. Besides, the implants inserted to replace the medial meniscus tended to protect the articular cartilage from increased stress after the removal of native medial meniscus. Similarly, in the study, although the analysis showed that the biomechanical properties of implants with cells were stiffer than that without cells, all of scaffolds are inferior to the native in biomechanics. With the respect to the chondronprotective effects, implants with seeded cells showed better results than that of cell free ones at each time point. However, we have to acknowledge that cartilage degradation was not totally avoided. 265 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 5. Macroscopic observations of joints in 24 weeks post operation. Meniscus tissue excised from the tibial plateau are shown on the right. Scale bar represented 10 mm.

Figure 6. Scanning Electron Microscope images of articular cartilage surfaces in femoral condyle and tibial plateau, Scale bar = 5 μm. The biomechanical properties of native meniscus are essential for the distribution of the stress or load on the articular cartilage, but the transplanted scaffolds with lower biomechanics are inferior to the native meniscus. Therefore, next endeavor should be made to generate truly bionic tissue-engineered meniscus with better microstructures and biomechanics by making full use of 3D printing with biocompatible materials. To our limited knowledge, this was the first study evaluating the effect on chondroprotection of 3D PCL scaffold seeded with bone marrow derived mesenchymal stem cells, which provided a good prospect that 266 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

the 3D PCL/stem cells composite scaffold might be a promising alternative for meniscus replacement.


Thermogel-Coated PCL Composite Scaffold for Enhanced Cartilage Tissue Engineering In addition to the meniscal regeneration, we also carried out a study in vitro on the regenerative cartilage augmented by the 3D-printed PCL scaffolds coated by the thermolgel (54). The aim of this study was to compare the proliferation, survival, and chondrogenic capacities of bone marrow mesenchymal stem cells in thermogel, PCL scaffold, and PCL/Gel scaffold in vitro. In this experiment, the 3D PCL scaffold was also fabricated by FDM techniques, which was then integrated with the polylactide (PLA), polyglycolide (PGA), and polyethyleneglycol (PEG) to form a composite scaffold for tissue-engineered cartilage. During the process of generating composite scaffold, we coated the surface of porous PCL backbone by submerging it into the aqueous solution of PLGA–PEG–PLGA copolymer. The harvested stem cells were seeded onto the PCL/Gel composite scaffold, or single thermogel or single PCL scaffold. The results showed that the elastic moduli were no significant differences among the osteochondral plug, single PCL scaffold, thermolgel coated PCL composite scaffold, while thermolgel revealed that the weakest mechanical properties, which was negligible. Compared with the cells seeded onto the PCL scaffold, cells onto the composite scaffold revealed a chondrocyte-like round morphology and were distributed evenly into the pores. The number of cells increased sharply on day 14 onto the composite scaffold, though the number of cells on day 7 in the composite scaffold and thermolgel are not significantly different from that on day 1, respectively. In contrast, cells in the PCL scaffold showed proliferative tendency after 1 day, however, the number of cells in PCL was exceeded by that in thermolgel-coated scaffold. The analysis of the matrix production on scaffold showed that the increasing GAG contents were detected in all groups, after culture in the chondrogenic medium for 7 days. On day 21, the more GAG contents were found in thermolgel and composite scaffold. Finally, we analyzed the expression of cartilage-specific gene. The level of Col II and AGC gene expression in PCL group was lower than those in the thermogel and composite scaffold groups at 10 days. There were the highest levels of the Col II and AGC expression in the thermogel and composite scaffold groups on day 21, whereas the highest amount of Col I expression was detected in the PCL scaffold group. The ALP expression level was not different among all the groups. As mentioned above, an ideal scaffold for tissue engineering needs sufficient mechanical strength and good biocompatibility as well. In spite of ease of use, the PLGA–PEG–PLGA copolymers are not enough stiff to withstand the mechanics, especially in a weight-bearing joint, like knee joint. Therefore, the application of this thermolgel in tissue-engineered cartilage has been curbed. PCL scaffold presents adequate mechanical properties, however, a sub-optimal structure for cell migration and spatial organization. In this work, we create a composite scaffold with the combination of PCL and thermolgel to exert their respective advantages. After coating the PCL with thermolgel, the composite scaffold was 267 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


obtained and the elastic moduli was similar to that of native osteochondral plug of rabbits in elastic moduli. Thermogel of the composite scaffold provided a better microenvironment than that of single PCL scaffold in terms of cell distribution and survival, and at the same time, mechanical properties of composite scaffold have been improved. The design concept of the thermolgel-PCL biphasic biomimetic scaffold has been verified in the preliminary study, indicating the potential of the scaffolds used for the cartilage regeneration. Next, it should be validated in further animal studies. Furthermore, 3D printing technology in future may develop the new strategy for fabricating the biphasic scaffold directly with the scaffold more precisely-controlled.

Current Issues and Future Prospects of 3D Printing for Meniscal and Cartilaginous Regeneration Since the recent decades, 3D printing technique has been given such a wide attention and a heated scientific topic, almost concerned with all of disciplines of medicine. Traditional concepts of medicine are indeed being challenged and revolutionized enormously with the rapid development of this technique. It is the innovative tool that brings new insights into tough medical problems for doctors and researchers. Presently, the technique of layer-by-layer directed by 3D printer for fabricating the cell and scaffold composites have shown the great potential of building complex 3D structures. Although meniscal and cartilaginous disorders have always been challenges in sports medicine in which to date many approaches fail to provide satisfactory result, tissue engineering technique augmented by 3D printing has shown the great potential to enhance the regeneration of menisci and cartilages. Some established regenerative approaches or tissue-engineered strategies have succeeded in regenerating or creating the living meniscus-like or cartilage-like tissues. Therefore, the future approach of tissue engineering technique combined with advanced 3D printing may create a new solution for regenerating complete and functional meniscus and cartilage. In our first research, we designed and printed the 3D PCL meniscal implants with three distinct mean pore sizes by FDM in order to explore the microstructures of scaffolds for the regeneration of meniscal tissues. As anticipated, the mean pore size of scaffold exerts effects on the regeneration of meniscal tissues in vitro or in vivo (53). Fabricating the microstructures of meniscal scaffold precisely makes it possible to explore the effect of mean pore size on cellular behaviors, which is a strength of 3D printing technique. In the second research, the similar 3D PCL scaffolds were also fabricated by FDM according to the pre-determined 3D model created by Mimics software, for the shape and size of 3D meniscal scaffold could be made as similar to the native meniscus as possible. Hence, there is another advantage of 3D printing technique: the bulk geometric shape of native meniscus or other tissues can be simulated to create a scaffold in case of mismatch. Overall, when integrating into the traditional tissue-engineered technology, the modern 3D printing technology drives the great improvement of tissue-engineered meniscus and cartilage as shown in the aforementioned work. 268 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Recently, bioprinting has emerged as an alternative for traditional regenerative methods, which has become the new generation of 3D printing technology for furthering the regenerative medicine. The definition of bioprinting can be described as an innovative technology that allows for the generation of highly arranged 3D tissue constructs via a layer-by-layer deposition process that combines cells and biomaterials in an ordered, precisely controlled, and predetermined way (55–57). Bioprinting of scaffolds and cells is becoming an effective approach to rebuild or simulate the microphysical environment and the relationship between cells, their ECM and local structures. Although, bioprinting technology has a good potential in 3D tissue engineering, in terms of architectures, compositions, and biological functions, the present bioprinted constructs are relatively simple. Especially, when it comes to bioprinted soft tissues, containing live cells within the construct, there has many challenges that need to deal with and this technique is still at very early stage. One of the great challenges is the unavailable results of long-term stability of the engineered tissue. Therefore, it is far too early to create regenerated meniscus and cartilage in clinical use by 3D printing or bioprinting. Although endeavor has been made to manipulate the ingrowth and integration of neo-tissues in the engineered process by recapitulating the microstructures and microenvironment of native meniscus or cartilage, it has been shown that the formation of neo-tissues inferior to the native ones in mechanical properties and structures (53, 54). The heterogeneity of meniscus and articular cartilage has been reported and focused by many researches (53, 57), and the heterogeneous structures are very hard to be constructed by current techniques in vitro, in spite of the much more precisely manufactural structures provided by the advanced 3D printing technology. Besides, each of bioprinting techniques has its own benefits and drawbacks. If the complex meniscal or cartilaginous tissue is needed to be fabricated, different printing techniques need be combined to overcome printers’ specific disadvantages to obtain complex 3D tissues. The integrating multiple techniques are able to be used to cope with various biomaterials with distinctive properties such as viscosity, sensitivity to pressure or gelling mechanism (48). Biomaterials for fabricating scaffolds support and maintain 3D tissues, which are crucial to tissue engineering directed by bioprinting technique. However, Among the present materials available, there are very limited ones which can be used for bioprinting. New printable biomaterials should be developed for meeting the following requirements: (1) support easy manipulation by bioprinting technique, (2) the structural integrity and compatibility with cells or tissues should be maintained during gelation/crosslinking and later tissue culture (33, 48). Compared with hard materials, materials for the fabrication of scaffold regenerating soft tissues are less available because of their poor mechanical properties (48). For instance, the fabricated constructs from hydrogel materials are very fragile and their poor mechanical strength are not capable of withstanding the surgical manipulation and high load after being implanted in vivo. Considering the key role of mechanical properties of meniscus and articular cartilage in knee joint, there is an urgent need to develop soft biomaterials for regenerating meniscus or cartilage, which can not only be printed with live cells together, but 269 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


provide satisfactory mechanical properties for further handling and withstanding mechanical load in vivo. Both of meniscal and cartilaginous tissues have the complex and anisotropy extracellular structures and thus, it is very difficult to fully mimic the native ECM compositions and structures for current materials to form the constructs possessing complex combinations and gradients (33). Therefore, the comprehensive understanding of extracellular microenvironment in target tissues need further improvement. Also, the relationship between cells and ECM will be required to be illuminated in further studies in order to mimic the model of interactions of cells and ECM. Overall, this is a very exciting time where the multidisciplinary collaborations are furthering apply 3D bioprinting for the field of regenerative medicine. In the field of meniscal and cartilaginous regeneration, future 3D printing technique or bioprinting technique may be an innovative and promising approach to enabling an efficient strategy for creating patient-specific meniscus and cartilage rapidly. Despite of many existing challenges and obstacles, the good prospects and possibilities that bioprinting combined with new biomaterials bring to tissue-engineered meniscus and cartilage are endless.

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Three-Dimensional Printing Technology Combined with Materials

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