Xenograft of bio-3D printed scaffold-free cartilage constructs derived from human iPSCs to regenerate articular cartilage in immunodeficient pigs

doi:10.21203/rs.3.rs-4615138/v1

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Xenograft of bio-3D printed scaffold-free cartilage constructs derived from human iPSCs to regenerate articular cartilage in immunodeficient pigs

https://doi.org/10.21203/rs.3.rs-4615138/v1

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Arthroplasty is currently the only option for reconstruction of large articular cartilage defects, mainly due to osteoarthritis. However, reconstruction with artificial materials has several issues such as deterioration, foreign body reaction, and bacterial infection. This study established a new method for articular cartilage reconstruction that fundamentally solves the problems associated with artificial materials by creating scaffold-free cartilage constructs and implanting them into extensive osteochondral defects without artificial materials. Tubular cartilage constructs were fabricated using a completely scaffold-free Kenzan bio-three-dimensional printing method with chondrogenic spheroids generated from human induced pluripotent stem cell-derived mesenchymal stem/stromal cells (iPSC-MSCs). The constructs were partially cut open to form a patch and implanted into osteochondral defects in the femoral trochlear groove of immunodeficient miniature pigs. The cartilage constructs were elastic and easy to handle, and abundant glycosaminoglycans and collagens were observed in the grafted site after implantation as well as in the articular cartilage. Cells at this site were positive forhuman vimentin, indicating that the cartilage constructs were successfully engrafted onto the host subchondral bone. Scaffold-free human iPSC-MSC-derived cartilage constructs implanted into osteochondral defects contributed to the regeneration of extensive osteochondral defects in the absence of artificial materials.

Biological sciences/Stem cells/Pluripotent stem cells/Induced pluripotent stem cells

Health sciences/Medical research/Pre clinical studies

cartilage regeneration

xenograft

bio-3D printer

scaffold-free

tissue engineering

iPSC

articular cartilage

Articular cartilage (AC), also known as hyaline cartilage, consists of chondrocytes and an extracellular matrix (ECM) composed mainly of type II collagen (Col II) and proteoglycans and covers the end of the bone in a joint. Because AC is a non-vascularized tissue with poor self-healing properties, it is repaired by fibrocartilage when damaged. However, fibrocartilage is mechanically inferior to hyaline cartilage; thus, the damaged AC will eventually deteriorate¹. Osteoarthritis (OA) is a major joint disease that affects numerous middle-aged and older people worldwide, causing chronic pain and loss of mobility². Eventually, in the late stages of OA, AC loss is extensive. In young individuals, trauma-induced AC damage can be treated with deliberate microfractures, which result in fibrocartilage coverage of the repaired tissue. At the expense of mechanical strength, this approach provides primary pain relief and the restoration of joint function³. However, professional athletes who received microfracture treatment showed worse performance and poorer post-injury outcomes than pre-injury⁴.

As cartilage damage does not heal spontaneously, cartilage regeneration has been studied; however, extensive cartilage defects in OA have not yet been regenerated. Currently, metal-based prostheses are the first choice for end-stage OA; however, various problems, such as infection and loosening, make their use in younger patients difficult⁵. Experimental attempts have been made to create anatomical cartilage implants using various materials as scaffolds⁶. However, scaffold materials, as well as metals, are prone to infection and elicit an immunological response⁷.

Therefore, we fabricated cartilage constructs using only cells. We developed a bio-three-dimensional (3D) printer based on the Kenzan method, which can fabricate arbitrarily shaped 3D cellular constructs without scaffolds^8,9,10. When spheroids consisting of tens of thousands of cells are stacked on microneedles and cultured, adjacent spheroids adhere and fuse to form a scaffold-free 3D construct. This construct avoids the issues associated with the use of artificial materials, as mentioned above. This bio-3D printing technology has been successfully used to fabricate a number of different tissues, including blood vessels¹¹, nerve conduits¹², trachea¹³, myocardium¹⁴, and liver¹⁵, with a wide variety of cell types. We previously reported the fabrication of human induced pluripotent stem cell (iPSC)-derived cartilage constructs in vitro¹⁶. iPSCs can be generated by adding four reprogramming factors^17,18 and have pluripotent capacity and self-renewal, and unlike embryonic stem cells, which must be harvested from embryos, there are no ethical issues associated with their use. However, while research into regenerative medicine using stem cells such as iPSCs is advancing dramatically, the clinical application of human iPSC-derived AC is still a long way off.

For the clinical application of tissue-engineered AC, it is necessary to conduct studies in large animal models that are similar in size and loading environment to humans rather than in small animals such as rats and rabbits. Pigs are diurnal and their knees are extended and loaded during the day. However, when they sleep, their knee joints are not loaded. This lifestyle is similar to that of humans; therefore, pigs have been considered a suitable animal model for AC implantation^19,20. Miniature pig (mini-pig) cartilage is histologically similar to human cartilage, with knee joint size, loading, lack of spontaneous damage healing, and collagen network arrangement similar to that of human joints^{21,22,23,24,25}. Furthermore, mini-pig knee joints have been shown to have stress values similar to those of human knee joints²¹. Itoh et al. created an immunodeficient mini-pig model to implant human dermal fibroblast-derived blood vessels for artery-vein bypass using the aforementioned bio-3D printer²⁶. This immunodeficient model is desirable for preclinical studies, in which human-derived cells are implanted to evaluate their long-term function.

In the present study, we implanted bio-3D printed human iPSC-derived cartilage constructs into extensive osteochondral defects in immunodeficient mini-pigs to evaluate the utility of this new alternative treatment for AC regeneration.

Fabrication of constructs

Using a bio-3D printer, spheroids were stacked on needle arrays and matured in a reactor with a reflux function for 30–76 days, resulting in the formation of tubular constructs 7 mm in diameter and 2 cm in length (Fig. 1a-c). After removal from the needle array, the tube shape was maintained by fusion of the spheroids; it was elastic and could be easily grasped without breaking (Supplementary Video 1). Histologically, numerous chondrocytes were observed, and the surrounding ECM area produced abundant glycosaminoglycans (GAGs), which were stained with safranin O and collagen fibers that were positive for both type I collagen (Col I) and Col II. Human-specific anti-vimentin antibody staining was strongly positive, in accordance with the cellular localization (Fig. 1d-g).

Results of implanting the cartilage constructs on osteochondral defects in immunodeficient pigs

Gross assessments

At one month in the implanted group, the osteochondral defect had become grossly filled with regenerated tissue and more than half of the depth had regenerated. White regenerated tissue was observed in the center of the defect, the margins of the defect were repaired with reddish tissue, and most of the borders were integrated (Fig. 2a). Three months after implantation, the entire defect was almost filled with white regenerated tissue and the borders were mostly integrated and obscured (Fig. 2b). In the control group at one month postoperatively, the defect was partially covered by white fibrous tissue, but the degree of defect repair was poorer than that in the implanted group, and approximately half of the edge had not been integrated (Fig. 2c). In the control group at three months, the border was generally clear, although it was almost entirely filled with white repaired tissue (Fig. 2d). In all groups, the macroscopic appearance revealed small cracks and fissures on the surface of repaired tissue. Therefore, only the mean score of the implanted group at three months postoperatively was “near normal,” while the rest was “abnormal” according to the International Cartilage Repair Society (ICRS) overall score (Fig. 3).

Histological and immunohistochemical assessments

One month postoperatively in the implanted group, abundant chondrocytes were identified by hematoxylin-eosin staining and further stained with human vimentin, which showed staining findings in accordance with cell localization, indicating that the chondrocytes were derived from human induced pluripotent stem cell-derived mesenchymal stem/stromal cells (iPSC-MSCs). The ECM surrounding the cells was mostly covered by thick GAG-rich AC-like tissue, and abundant Col I- and Col II-positive collagen fibers were observed (Fig. 4a). In addition, three months after implantation, an extensive layer of cartilage positively stained with human vimentin and safranin O was observed. Collagen fibers were positive for both Col I and Col II, but Col I stained more lightly at three months than at one month postoperatively (Fig. 4b). In the control group, one month postoperatively, most of the repaired tissue was Col I-positive and covered by a thick fibrocartilaginous layer without GAGs, and the cells were vimentin-negative (Fig. 4c). At three months postoperatively, the majority of the cartilage was covered with fibrocartilage, although there was a small internal layer of cartilage that was positively stained with safranin O and Col II (Fig. 4d). Chondrocytes in the implanted group were arranged in an orderly manner perpendicular to the articular surface and in layers, similar to the normal AC, and were tightly connected to the host subchondral bone in the deeper layers, creating a solid structure (Supplementary Fig. 1). The implanted groups had a greater area that was positively stained with safranin O and tended to have higher hyaline cartilage scores than those of the control group (Fig. 5).

Mechanical assessments

The compressive strength was 42.37 ± 14.39 MPa for the postimplanted cartilage and 63.65 ± 25.4 MPa for the native AC. The strength of cartilage constructs before implantation was 0.18 ± 0.05 MPa. The strength of the post-implanted AC was significantly higher than that of the constructs (P < 0.01) (Fig. 6).

In the present study, patch-formed cartilage constructs were implanted into extensive osteochondral defects created in the femoral trochlear groove of mini-pigs. Implanted human iPSC-derived cartilage constructs matured without rejection in immunodeficient mini-pigs, and AC-like tissue was confirmed to be donor-derived by human-specific anti-vimentin antibody staining. Histological examination of the constructs showed many chondrocytes and a GAG-rich ECM, indicating that the cartilage properties were maintained and matured after implantation. The scaffold-free cartilage constructs were strong enough to endure handling during the surgery. Furthermore, the implanted constructs were significantly stronger than before implantation, and their compressive strength increased to a value approximating that of normal cartilage within three months, indicating that maturation also proceeds in vivo. Although several cartilage regeneration studies are currently underway, the extent of cartilage regeneration remains limited. Autologous chondrocyte implantation, which is indicated for traumatic cartilage injuries with relatively large defects, involves harvesting and culturing chondrocytes from normal cartilage and seeding them onto scaffold material. This treatment requires the sacrifice of cartilage tissue and two surgeries, and dedifferentiation in expanded cultures remains an issue²⁷.

To create cartilage constructs using bio-3D printing, it is necessary to select appropriate cells. In chondrogenic differentiation, iPSC-MSCs have higher expression of SOX9, COL2A1, and Aggrecan, and lower expression of hypertrophic markers, such as COL10A1 and RUNX2, than human bone marrow-derived mesenchymal stem/stromal cells (BM-MSCs), indicating that they are a better source for AC regeneration than human somatic stem cells²⁸. Cartilage derived from primary chondrocytes and BM-MSCs produced less ECM than iPSC-derived cartilage^16,29. Somatic stem cells, also called MSCs, have a multipotent differentiation potential, but several issues prevent their widespread use. For example, they must be harvested from healthy tissues, the differentiated cartilage tends to be hypertrophic, and their proliferative differentiation potential decreases with age^30,31. In addition, MSCs suffer from chromosomal aberrations and deterioration after repeated passages, making it difficult to obtain a large number of high-quality cells for clinical use in expanded culture³². However, human iPSC-derived neural crest cells (iNCCs) can be cryostocked³³ and differentiate into iPSC-MSCs to produce large quantities of cartilage spheroids of uniform quality and size, making them superior for bio-3D printing¹⁶. While, there was a previous report in which iPSC-derived cartilage spheroids were produced not through iNCCs, but microlevel size control and mass production may be difficult to achieve by using the protocols³⁴.

Although iPSCs have been reported to be associated with a risk of tumorigenesis owing to their pluripotency^35,36,37, the protocol for inducing chondrogenesis from iPSC-MSCs derived from iNCCs showed excellent chondrogenic differentiation without tumor formation or upregulation of pluripotency markers³⁸. In the present study, cartilage constructs fabricated following the protocol showed no malignant cells, and a Col II-positive GAG-rich ECM was observed around abundant chondrocytes. During the remodeling process associated with the maturation of cartilage ECM, Col I gradually disappears from the ECM and is replaced by Col II³⁹. The constructs in the present study were positive for both Col I and Col II; however, Col I levels decreased, whereas Col II levels increased over time, as described in a previous report¹⁶. Further long-term evaluations of this maturation process are needed, including assessments of the ratios of Col I and Col II, and whether there is a trend toward hypertrophy, as with other MSCs.

In previous studies, undifferentiated MSCs have been implanted into osteochondral defects and regenerated hyaline cartilage^20,40. In the present cartilage constructs, it was expected that poorly differentiated constructs would similarly differentiate into hyaline cartilage after implantation; however, this was not the case. Cells that did not differentiate into chondrocytes prior to implantation may not have been undifferentiated iPSC-MSCs, but instead may have differentiated into other cells or reached cellular senescence (Supplementary Fig. 2a-c). In addition, because tissue-engineered constructs seeded with undifferentiated iPSC-MSCs do not differentiate into chondrocytes when implanted³⁸, it may be necessary to differentiate iPSC-MSCs into cartilage before implantation. After differentiating iPSC-MSCs into cartilage, we fabricated 3D cartilage constructs using a Kenzan bio-3D printer in the present study. However, several issues have arisen in this regard. The yield rate of the cartilage constructs was unsatisfactory; 8 out of 12 constructs produced abundant GAG in most regions; therefore, the fabrication protocols might have to be improved. Of note, attempts have been made to refine currently available protocols for producing high-quality chondrogenic spheroids⁴¹. In addition, the edges of the implanted construct, which had inappropriate thickness, were shaved by friction with the contralateral patella (Supplementary Fig. 3a-b). However, the appropriate cartilage construct regenerated the defects without shaving (Supplementary Fig. 3c-e), suggesting that we must improve not only the construct fabrication but also the surgical protocols, including the suture technique, external fixation, and duration of unloading.

Xenografts of bio-3D printed scaffold-free cartilage constructs derived from human iPSC-MSCs have shown cartilage regeneration in extensive osteochondral defects. Therefore, cartilage constructs may be suitable for extensive regeneration of AC. However, the present study had several limitations that should be mentioned, including the small number of animals and the fact that statistical analyses were not performed. In the future, the number of animals should be increased, and histological, imaging, and physical activity evaluations should be conducted over a longer period.

The Kenzan bio-3D printing method can fabricate constructs with complex shapes using computer-aided design (CAD) data of articular surface shapes¹⁶. If the fabrication protocol could be further improved to produce high-strength cartilage constructs, a surface-replacing cellular joint implant could be realized (Supplementary Fig. 4, Supplementary Video 2).

Cell culture and MSC induction

Human iNCCs differentiated from human iPSC line 414C2 were used in this study. Cultures and passages were performed as previously described^16,38. Briefly, iNCCs were grown in chemically defined medium (CDM) consisting of Iscove's modified Dulbecco's medium (Sigma-Aldrich, St. Louis, MO, USA) and Ham’s F-12 (Gibco, Grand Island, NY, USA) at a 1:1 ratio. CDM contained 1% chemically defined lipid concentrate (Gibco), 15 µg/mL apo-transferrin (Sigma-Aldrich), 450 mM monothioglycerol (Sigma-Aldrich), 5 mg/mL purified bovine serum albumin (BSA; Sigma-Aldrich), 7 µg/mL insulin (Wako Pure Chemical Industries, Osaka, Japan), 10 µM SB431542 inhibitor (Selleck Chemicals, Houston TX, USA), 20 ng/mL epidermal growth factor (EGF; R&D Systems, Minneapolis, MN, USA), 20 ng/mL basic fibroblast growth factor (bFGF; Wako), and a supplement containing 50 U/ml penicillin and 50 µg/ml streptomycin (Invitrogen, Carlsbad, CA, USA). iNCCs were cultured on fibronectin-coated dishes (Millipore, Billerica, MA, USA) and Accutase (Wako) was used for passaging.

To differentiate iNCCs into iPSC-MSCs, CDM was replaced with MSC medium, which contained minimum essential medium Eagle alpha modification (aMEM; Nacalai Tesque Inc., Kyoto, Japan), 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 5 ng/mL bFGF (Wako), 50 U/ml penicillin, and 50 µg/ml streptomycin. Trypsin-EDTA (0.25%; Gibco) was used to passage cells. The medium was changed every other day.

Chondrogenic induction and preparation of spheroids

As previously reported¹⁶, chondrogenic induction was performed using iPSC-MSCs from passage three. Chondrogenic Differentiation Basal Medium supplemented with hMSC chondrogenic single quotes containing dexamethasone, ascorbic acid, ITS + supplement, GA-1000, sodium pyruvate, proline, and L-glutamine (Lonza, Walkersville, MD, USA) was used for chondrogenic differentiation. Additionally, 40 ng/ml platelet-derived growth factor-BB (PDGF-BB; R&D Systems) was used from days 0 to 10 after the start of differentiation induction, and 10 ng/ml transforming growth factor beta-3 (TGF-β3; Peprotech Inc., Rockey Hill, NJ, USA) and 50 ng/ml bone morphogenetic protein 4 (BMP4; R&D Systems) were added from days 6 and 10, respectively. From the start of induction, 4 × 10⁴ cells were seeded in 96-well non-adherent plates (Sumitomo Bakelite Co., Ltd., Tokyo, Japan), and spheroids reached a diameter of approximately 700 µm after 10 days. The medium was changed twice per week. All the cultures were grown in a humid environment at 37°C and 5% CO₂.

Biofabrication of cartilage constructs

After spheroid-induced chondrogenic differentiation for 10 days in a 96-well plate, the spheroids were placed on a 7-mm-diameter Kenzan with a circular array of 36 microneedles using the Regenova Bio-3D Printer (Cyfuse Biomedical K.K., Tokyo, Japan) to create a 3D tube that had previously been designed using CAD. Regenova automatically picks up spheroids by suctioning them with a nozzle, and places them on the Kenzan, where a fine-needle array is arranged. The spheroids fuse with each other, and scaffold-free 3D constructs can be created^9,10. After stacking, the constructs were cultured in a bioreactor with an internal reflux function, and the chondrogenic differentiation induction medium was changed twice a week for over a month to allow sufficient maturation of the constructs for implantation.

Animals and creation of the operational immunodeficient pig (OIDP) model

Seven mini-pigs (Nippon Institute for Biological Science [NIBS]: 3 males, and Göttingen: 2 males and 2 females; Oriental Yeast Industry, Nagano, Japan) were used in this study. Their mean age and body weights were 23.8 (11–46) months old and 31.5 (18.2–40.6) kg, respectively. For surgical anesthesia, the mini-pigs were sedated with intramuscular injections of atropine sulfate (0.05 mg/0.1 ml/kg), medetomidine hydrochloride (0.05 mg/0.05 ml/kg), and midazolam (0.5 mg/0.1 ml/kg) followed by a mixture of N₂O:O₂ = 1:1 gas, and 2.0% general anesthesia was maintained with isoflurane and managed with a ventilator.

In accordance with previous reports²⁶, the three lobes of the thymus were excised after a midline incision in the ventral region of the neck. The spleen was removed through a midline abdominal incision. After the gastric corpus was exposed, a gastrostomy tube (GB gastrostomy balloon catheter, tubular type, cannula outer diameter: 24 Fr; Nipro Corporation, Osaka, Japan) was surgically inserted, and the insertion site was ligated with a drawstring suture and fixed to the abdominal cavity wall. One end of the tube was suture-fixed to the back skin through a subcutaneous tunnel. A central venous catheter (Medicat L CV-UK kit, single lumen type, catheter outer diameter: 14 G; Japan Covidien Corporation, Tokyo, Japan) was placed in the anterior vena cava sinus for blood sampling and sutured to the neck. The inside of the vascular catheter was locked with heparinized saline solution (200 units/ml) every 1–3 days.

Tacrolimus (0.25 mg/kg) and mycophenolate mofetil (30 mg/kg) were administered twice daily, and prednisolone (20 mg/body) was administered once daily through a gastrostomy tube for immunosuppression. Blood samples for tacrolimus and mycophenolate mofetil were collected twice a week, and the doses were adjusted to achieve trough blood levels of 5–10 ng/ml and 1–3 µg/ml, respectively.

Ethical review of all procedures in this study was conducted by the Institutional Animal Care and Use Committee and the Human Sample Experiments Ethical Committee of Nihon Bioresearch Inc. (Approval Nos. 380246, 390193, 409063, 409190, and 410042), and the Institute of Animal Experimentation, Kurume University School of Medicine (Approval No. 2022 − 175). We confirmed that all methods were carried out in accordance with relevant guidelines and regulations and reported in accordance with ARRIVE guidelines for the reporting of animal experiments.

Implantation of cartilage constructs into extensive AC defects

One week after OIDP creation, the same anesthesia was administered, and the knee joints were incised using a medial parapatellar approach to dislocate the patella and expose the trochlear groove. Osteochondral defects (20×10 mm, 2 mm deep) were created in the trochlear groove using a bone chisel, and mature cartilage constructs were cut into patches and implanted (Fig. 7a, Supplementary Video 3). The constructs were threaded with sutures (7 − 0 Proline; Ethicon, Somerville, NJ, USA) and fixed onto osteochondral defects with pull-out sutures (Fig. 7b, c). The spheroids of the constructs were well-fused to each other and fixed onto the defects using pull-out sutures without tearing. In the control group, only osteochondral defects of the same size as those in the implantation group were created. Four implanted knees and four control knees were created (two knees each at one and three months). In both the implanted and control groups, two pins were inserted into the femur and tibia, and an external fixator (IMEX SK Linear External Fixator Small; Kirican Ltd., Tokyo, Japan) was used to limit knee joint motion. Two weeks postoperatively, the external fixator was removed under sedation, and the knee joint was allowed to move freely. Ampicillin (1000 mg/5 ml/body) was administered to prevent postoperative infection, and buprenorphine (0.01 mg/0.05 ml/kg) was given for pain relief for up to 4 days after surgery. At 1 or 3 months postoperatively, the mini-pigs were sedated with intramuscular injections of medetomidine hydrochloride (0.05 mg/0.05 ml/kg) and midazolam (0.5 mg/0.1 ml/kg), deeply anesthetized with thiamylal sodium (25 mg/1 ml/kg) and euthanized by exsanguination via the femoral artery.

Gross evaluations and histological and immunohistochemical assessments

Gross findings were graded by two investigators using the ICRS scores (Table. 1). The dissected femoral patellar glenoid groove was fixed in 10% formalin (Wako Pure Chemical Industries). Longitudinal sections were cut parallel to the groove and embedded in paraffin, and 5-mm-thick sections were prepared. Tissues were stained with hematoxylin-eosin (HE; Merck, Darmstadt, Germany) and SOFG (0.1% Safranin-O; Nacalai Tesque Inc.; 0.05% Fast Green: Wako) for morphological analyses. ColI (Southern Biotech, Birmingham, AL, USA) and ColII (Southern Biotech) staining were performed to analyze collagen deposition. Human vimentin (anti-vimentin antibody [SP20] [ab16700]; Abcam, Cambridge, UK) staining was performed to assess whether the repaired tissue was derived from human iPSCs. Histological assessments were scored by two investigators using the modified O’Driscoll histologic scoring system⁴² (Table. 2). Sections of mature cartilage constructs were prepared in the same manner and were histologically evaluated.

Mechanical assessments

Compressive strength was measured using a Digital Force Gauge (ZTA-500 N; IMADA, Aichi, Japan). The post-implanted cartilage (n = 5) 3 months after implantation and native cartilage (n = 5) were trimmed to cylindrical shapes using a biopsy trepan (diameter: 2.0 mm) (Kai Industries, Gifu, Japan). Most of the subchondral bone was excised and measured by using a flat compression attachment. Before implantation, cartilage constructs (n = 6) were formed into the appropriate shapes and strength tested in the same manner.

Statistical analyses

All data are expressed as the mean ± standard deviation. Comparisons among the three groups for mechanical assessments were performed using one-way analysis of variance (ANOVA), followed by Bonferroni's multiple comparison test. Statistical significance was set at P < 0.05.

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Acknowledgments

This study was supported by the Incubation Program of Kyoto University and received funding from Healios K.K. in Japan.

iPSC-MSCs were successfully generated by S Tamaki and S Nagata at Kyoto University. This study was conducted at Nihon Bioresearch Inc. and the Center for Disease Model Research, Kurume University School of Medicine. Dr. Kobayashi demonstrated the surgical techniques of the immunodeficient mini-pig model. We express our deepest gratitude to Dr. Kobayashi, Dr. Shiozawa, Dr. Sakai, and the other staff of these institutions for their support.

Author contributions

T.N. performed the experiments, analyzed the data, and drafted the manuscript.

A.N. performed the experiments and analyzed the data.

D.M. implanted the cartilage constructs, interpreted the data, and edited the manuscript.

H.Y. implanted the constructs and performed gross and histological assessments.

S.K. implanted the constructs.

Y.N. provided advice on the manuscript.

D.Z., C.Z., Y.I.,M. Ikeya. and J.T. provided iNCCs derived from human iPSC line 414C2.

M. Itoh. developed the operational immunodeficient pig model.

M.M. provided advice on the research design and interpreted the data.

K.N. developed the bio-3D printer system, designed and supervised the experiments, interpreted the data, and edited the manuscript.

Competing interests

K.N. is a co-founder and shareholder of Cyfuse Biomedical KK, chief technical officer of Arktus Therapeutics, and an inventor/developer designated on the patent for the bio-3D printer. Patent title: Method for Production of Three-Dimensional Structure of Cells; patent number: JP4517125. Patent title: Cell structure production device; patent number: JP5896104. The other authors declare no competing interests regarding the publication of this article.

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Tables 1 to 2 are available in the Supplementary Files section

Competing interest reported. K.N. is a co-founder and shareholder of Cyfuse Biomedical KK, chief technical officer of Arktus Therapeutics, and an inventor/developer designated on the patent for the bio-3D printer. Patent title: Method for Production of Three-Dimensional Structure of Cells; patent number: JP4517125. Patent title: Cell structure production device; patent number: JP5896104. The other authors declare no competing interests regarding the publication of this article.

SupplementaryFigure.1.jpg
Supplementary Figure 1. The chondrocytes in the implanted group at one month postoperatively were arranged in an orderly manner perpendicular to the articular surface and in layers and were tightly connected to the host subchondral bone. Scale bar: 500 µm.
SupplementaryFigure.2.jpg
Supplementary Figure 2. The poorly differentiated cartilage constructs derived from human iPSC-MSCs. a: Gross image of the cartilage constructs. Histological evaluations of the cartilage constructs that were stained with (b) SOFG, (c) Col II. Scale bar: 1 mm in a, 500 µm in b-c.
SupplementaryFigure.3.jpg
Supplementary Figure 3. a: The fixed cartilage construct protruded too far outward. b: The edge of the implanted construct was shaved by friction with the contralateral patella. c: The construct with appropriate thickness fit onto the osteochondral defects. d: The appropriate cartilage construct was engrafted without loss and regenerated the defects. e: Side view of the same knee as in (d). Scale bar: 5 mm.
SupplementaryFigure.4.jpg
Supplementary Figure 4. a, b: The UKA model of a cartilage construct. Based on CAD data output from CT images, cartilage constructs with the articular surface shape of the knee joint were created using a bio-3D printer. Scale bar: 1 mm. UKA, unicompartmental knee arthroplasty; CAD, computer-aided design; CT, computed tomography.
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SupplementaryVideo1.mp4
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Tables.docx
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Xenograft of bio-3D printed scaffold-free cartilage constructs derived from human iPSCs to regenerate articular cartilage in immunodeficient pigs

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Abstract

Figures

Introduction

Results

Fabrication of constructs

Results of implanting the cartilage constructs on osteochondral defects in immunodeficient pigs

Gross assessments

Histological and immunohistochemical assessments

Mechanical assessments

Discussion

Material and methods

Cell culture and MSC induction

Chondrogenic induction and preparation of spheroids

Biofabrication of cartilage constructs

Animals and creation of the operational immunodeficient pig (OIDP) model

Implantation of cartilage constructs into extensive AC defects

Gross evaluations and histological and immunohistochemical assessments

Mechanical assessments

Statistical analyses

Declarations

References

Tables

Additional Declarations

Supplementary Files

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