Academic Editor: Piergiorgio Gentile
Background: Although autogenous bone implantation is considered to be
the gold standard for the reconstruction of bone defects, this approach remains
challenging when treating extensive bone defects (EBDs). Therefore, artificial
materials (AMs) such as artificial bone and scaffolds are often used for treating
EBDs. Nevertheless, complications such as material failure, foreign body
reaction, and infection are common. To overcome these issues, we aimed to develop
a new treatment for an EBD using scaffold-free adipose-derived stromal cells
(ADSCs) to fabricate chondrogenic/osteogenic-induced constructs without AMs.
Methods: ADSCs were obtained from the subcutaneous adipose tissue of
8-week-old female Wistar rats (n = 3) and assessed to determine their potential
for multilineage differentiation into adipocytes (Oil Red O staining),
chondrocytes (hematoxylin and eosin, Alcian blue, and Safranin O staining), and
osteoblasts (Alizarin red and von Kossa staining). Spheroids (n = 320), each
containing 3.0
As part of the skeletal system, the bone plays an important role in supporting the body. Extensive defects in the long bones are common orthopedic disorders following comminuted fractures [1, 2]. Such extensive bone defects (EBDs) are also often observed after surgical resection of tumors of the maxillofacial bone, as it is necessary to secure a surgical margin to completely remove the tumor [3]. Autogenous bone (AB) implantation is considered to be the gold standard for the treatment of EBDs [4]. However, this method is clinically challenging owing to the limitations of bone fixation methods and its success is dependent on the size of the bone graft [5]. Therefore, in recent years, artificial materials (AMs) such as artificial bones and scaffolds have been applied to the treatment of EBDs [6, 7, 8, 9, 10]. Nevertheless, AMs are sometimes inferior to AB in terms of strength and integration with the surrounding bone tissue [11]. Additionally, AMs often lead to problems such as a long-term foreign body reaction after implantation and biofilm formation following bacterial infection [12]. As AMs can also obstruct cell–cell adhesion and inhibit self-organization, it can take a long time to form mature and healthy bones [13]. A potentially effective and efficient approach to solve these problems could be to produce three-dimensional (3D) bone and cartilage-like tissues using only cells without any AMs.
Many types of stem cells have been studied in parallel with development of the regenerative medicine field [14, 15]. Mesenchymal stem cells, isolated from various tissues such as the bone marrow and adipose tissue, exhibit self-proliferation and multipotency properties [16, 17]. Among them, adipose-derived stromal cells (ADSCs) and stromal vascular fraction cells (SVFs) can differentiate into various cell types such as adipocytes, myoblasts, osteoblasts, chondrocytes, and neurons [18, 19, 20, 21, 22, 23, 24, 25, 26, 27]. Indeed, ADSCs and SVFs have already been used for regenerative medicine [28, 29]. In addition, by combining these cells with various biomaterials such as hyaluronic acid, platelet-rich plasma (PRP), and fat grafts, the range of application has been further expanded, and these cell types are thus expected to have further application prospects in the future [29, 30, 31, 32, 33, 34].
With the aim of developing a new bone reconstruction method for the treatment of EBDs, in this study, we used a bio-3D printer with a needle array to fabricate scaffold-free 3D bone-like and cartilage-like constructs using only ADSCs without AMs (Fig. 1).
Outline of this study. (A) Adipose tissue collection from rats. (B) Enzymatic treatment of adipose tissue. (C) Trilineage differentiation of adipose-derived stromal cells (ADSCs). (D) Spheroid formation of ADSCs. (E) Fabrication of a scaffold-free cellular construct using a 3D bioprinter. (F) Chondrogenic and osteogenic induction of the constructs.
Female Wistar rats (8 weeks old, n = 3; Charles River Laboratories Japan, Inc.,
Shiga, Japan) with a mean weight of 230 g (220–240 g) were used for the
isolation and expansion of ADSCs (Fig. 1A). All animals were housed under a 12-h
dark–light cycle (light from 08:00 to 20:00) at 23
Intrascapular adipose tissue (0.5–0.8 g per animal) was aseptically collected
under general anesthesia (induction anesthesia: 5% isoflurane, maintenance
anesthesia: 2.5% isoflurane; Fujifilm Wako Pure Chemical Industries, Ltd.,
Osaka, Japan), minced, and digested for 3 h in phosphate-buffered saline (PBS)
containing 0.1% collagenase (collagenase type I; Worthington Biochemical
Corporation, Lakewood, NJ, USA). The digests were filtered through sterile gauze
and centrifuged at 210
ADSCs collected from the subcutaneous adipose tissue of the intrascapular region of rats were differentiated into adipocytes, chondrocytes, and osteocytes (Fig. 1C).
The collected ADSCs were seeded at 2.0
Cell suspensions of 2.5
For osteogenic differentiation, ADSCs were seeded in a 6-well plate (Thermo
Fisher Scientific) with cell-CM at an initial density of 3.0
Two cell suspensions of 2.5
At least 9.6
Bio-3D printer with a needle array (Kenzan). (A) Bio-3D
printer. (B) Hollow 9
The Kenzan was placed in an original perfusion chamber, which can directly
supply the hollow structure with medium, and then cultured with the spheroids at
37
Fusion of two ADSC constructs. Two constructs were cultured side by side on a plastic tube to facilitate fusion.
Tensile tests were performed using a Tissue Puller™ device (DMT,
Ann Arbor, MI, USA) for three fused ADSC constructs. Small stainless pins served
as grips for the individual structures, after which the constructs were pulled in
tension to failure at a rate of 50
The prepared ADSC constructs were divided into a chondrogenic induction group
and a control group. The induction group comprised two subgroups: one in which
induction with CIM was started immediately after printing and another in which
induction was started when the construct was removed from the Kenzan 4 days after
printing. The control group was cultured in construct-CM. Culturing was performed
in the original bioreactor at 37
The prepared ADSC constructs were divided into an osteogenic induction group and
a control group. The induction group comprised two subgroups: one in which
induction with OIM was started immediately after printing and another in which
induction was started when the construct was removed from the Kenzan 4 days after
printing. The OIM was supplemented with additional factors (10 ng/mL
TGF-
The fabricated spheroids and constructs were imaged in 96-well plates using
Constructs were fixed in 10% neutral-buffered formalin for 15 days and embedded
in paraffin. Serial sections (7
Oil red O staining of the adipogenic-induced ADSCs showed reddish oil droplets in a greatly expanded cytoplasm (Fig. 4A). The cells differentiated very quickly (within 6 days) and produced oil droplets. The production of oil droplets indicates that the ADSCs differentiated into adipocytes. In uninduced ADSCs, no oil droplet production was observed and staining was negative (Fig. 4B).
Adipogenic, chondrogenic, and osteogenic differentiation of
ADSCs. (A,B) Oil red O staining of induced (A) and uninduced control (B) ADSCs.
Scale bar = 50
After chondrogenic induction of ADSC spheroids, large oval cells were observed with an Alcian blue- and Safranin O-stained cartilage matrix surrounding the cells (Fig. 4C–E). In contrast, the uninduced spheroids showed no large oval cells and the staining was negative (Fig. 4F–H).
For osteogenic induction of ADSCs, the entire basal surface of the well surrounding the cells was strongly stained with Alizarin red (Fig. 4I), whereas uninduced ADSCs were negative for Alizarin red staining (Fig. 4J). After osteogenic induction and staining of spheroids, whole spheroids were found to be positively stained with Alizarin red (Fig. 4L), while von Kossa staining confirmed the presence of calcium throughout the spheroid (Fig. 4M); in contrast, the uninduced spheroids were negative for Alizarin red and von Kossa staining (Fig. 4O,P).
The load, strength, and stiffness of the three fused ADSC constructs were
measured (Fig. 5). The calculated values were as follows: load, 195.3
Fabrication of fused ADSC constructs. (A) The spheroids were arranged on the needles of the Kenzan as per the computer design (day 0). (B) After 5 days of incubation, the spheroids fused together to form a single construct (day 5). (C) The two constructs were removed from the Kenzan and placed side by side on a plastic tube (day 5). (D) Two constructs fused together in maturation culture (day 15; 5 days on Kenzan and 10 days on the plastic tube). (E) Constructs that have undergone further incubation in the maturation culture (day 28; 5 days on Kenzan and 23 days on the plastic tube). Scale bar = 1 mm.
Osteogenic-induced spheroids were subjected to
The two induced and control constructs were subjected to
Macroscopic and
HE staining of the uninduced constructs showed that the boundaries between the spheroids were blurred and that the spheroids were fused into a single construct. In addition, nuclei were found distributed throughout the structure, confirming that the cells were evenly distributed (Fig. 8A). Alcian blue, Alizarin red, von Kossa, and Safranin O staining were negative, and no cartilage matrix was observed (Fig. 8B–E).
Histology of the uninduced and induced constructs. (A–E) HE,
Alcian blue, Safranin O, Alizarin red, and von Kossa staining of the uninduced
constructs. HE, Alcian blue, Safranin O, Alizarin red, and von Kossa staining of
the day 0 (F–J) and day 4 (K–O) chondrogenic-induced constructs. HE, Alcian
blue, Safranin O, Alizarin red, and von Kossa staining of the day 0 (P–T) and
day 4 (U–Y) osteogenic-induced constructs. Scale bar = 500
For both the day 0 and day 4 chondrogenic-induced constructs, large oval cells were observed whose nuclei were strongly stained with HE, similar to the results of the chondrogenic-induced spheroids (Fig. 8F,K). Alcian blue staining indicated the presence of an abundant cartilage matrix around the cells (Fig. 8G,L), which was also positively stained with Safranin O (Fig. 8H,M). The constructs were negative for Alizarin red and von Kossa staining, and no calcium production was observed (Fig. 8I,J,N,O). In the day 0 construct, chondrogenic differentiation was uniformly observed along the periphery of the constructs, and a layered structure was formed (Fig. 8F,G,H). In contrast, the day 4 structures showed no regularity in the areas of chondrogenic differentiation and fewer areas were stained more darkly with Alcian blue and Safranin O compared with the staining of the day 0 construct (Fig. 8L,M). Thus, the day 0 construct showed more pronounced chondrogenic differentiation than the day 4 construct. In common with both the day 0 and day 4 structures, chondrogenic differentiation was relatively poor inside the structures in contact with the plastic tube.
Histological specimens of the osteogenic-induced constructs showed areas that were simultaneously stained with HE, Alizarin red, and von Kossa, indicating calcium deposition (Fig. 8P,S,T,U,X,Y). In contrast, Alcian blue and Safranin O staining were negative (Fig. 8Q,R,V,W), and no cartilage matrix was observed. There was no substantial difference noted in the areas stained with Alizarin red and von Kossa stain between the day 0 and day 4 constructs. With respect to the distribution of calcified areas, most of the intensely stained areas in the day 0 constructs were found inside the constructs, whereas most of these areas were observed at the periphery of the day 4 constructs.
In this study, we successfully fabricated the first scaffold-free bone-like constructs and cartilage-like constructs using rat ADSCs. In particular, reconstruction of the bone tissue with sufficient strength in vitro has not been achieved to date. Therefore, we believe that one effective approach would be to implant our scaffold-free ADSC constructs in an uninduced state and allow them to mature into bone tissue in vivo. Furthermore, we confirmed that the two constructs could fuse together. By fusing constructs, the size and shape of the constructs that can be fabricated becomes more diverse. The results of the trilineage differentiation experiments indicated that ADSCs have the ability to differentiate not only into adipose tissue but also into bone and cartilage.
The strength test showed similar results (39.1
Notably, there was a marked difference in the
Comparison of the histological evaluation of uninduced, chondrogenic-induced, and osteogenic-induced constructs showed that ADSCs maintain their differentiation potential after forming constructs. Chondrogenic-induced constructs showed more pronounced chondrogenic differentiation at day 0 compared with that of the day 4 constructs. The cartilage matrix is produced by chondrocytes and is rich in type II collagen fibers [41]. It is presumed that chondrogenic differentiation is promoted by starting the induction process immediately after printing, which results in the production of a large amount of ECM around the cells, as a characteristic of cartilage tissue such as type Ⅱ collagen fibers. It is possible that during spheroid fusion—before induction—of the day 4 constructs, cellular adhesion was instead enhanced, and the ECM components specific to the cartilage tissue, such as proteoglycans, could not be produced around the cells, or other types of collagen fibers such as type I collagen, which is not specific to cartilage tissue, were produced. This is consistent with our previous study on cartilage constructs using induced pluripotent stem cells (iPSCs), which showed that the timing of additive factors for induction is very important [41]. The scaffold-free ADSC constructs prepared in this study were found to be as strong as soft tissues such as the muscle tissue and thyroid gland in the uninduced state, and we assume that the induced constructs have the same or greater strength. To our knowledge, there are no reports of scaffold-free 3D cartilage constructs fabricated using only ADSCs, which have advantages over smaller constructs such as micromasses or spheroids. This is one of the main new achievements in this study. In our previous study, we found that both bone and cartilage were regenerated after implantation of ADSC constructs into osteochondral defects in the knee joints [42, 43]. Another report demonstrated the formation of hyaline cartilage after subcutaneous implantation of fabricated cartilage pellets, although these were derived from iPSCs [44]. Our cartilage-like construct is expected to have application in the reconstruction of bone defects through the process of endochondral ossification after implantation.
Histological evaluation of the osteogenic-induced constructs showed strong staining with Alizarin Red and von Kossa, which was inconsistent with the results of CT imaging, indicating that calcium salt deposition by tissue staining does not correlate with CT imaging results. For histological evaluation of bone-like constructs, the presence of calcification alone is not sufficient. However, if the construct can be detected in CT images, it can be assumed that the crystal structure is at least sufficiently dense to not be penetrated by X-rays. There have been several reports on the fabrication of bone-like constructs using scaffolds [36, 45, 46]; however, producing calcified scaffold-free 3D constructs with CT values comparable to those of normal cortical bone in vitro is unprecedented. Furthermore, there is no previous description of the strength of bone-like tissue constructs fabricated using existing scaffolds, and it remains questionable whether structures with strength comparable to that of bone tissue can be fabricated. Based on existing reports, the fabrication of bone-like constructs in vitro remains a very challenging task. Moreover, we were unable to perform compression tests in this study because we could not achieve calcification of the entire structure; nevertheless, the fact that we were able to produce calcified 3D constructs with CT values comparable to cortical bone using only ADSCs is considered to be a major achievement. The final goal of this study was to develop an alternative clinical technique for AB implantation. For this purpose, it was necessary to fabricate strong constructs. It has been reported that osteogenesis of the bone tissue is accelerated by mechanical stimulation [47]; however, we did not mechanically stimulate the constructs, as we only employed perfusion culture. It is thus possible that the lack of mechanical stimulation may have contributed to the partial calcification and insufficient construct strength. In the future, the effects of incubation with mechanical loading should also be investigated.
There are also reports of improved wound healing [31, 32] and accelerated angiogenesis [33] when ADSCs were used in combination with hyaluronic acid or PRP; thus, these biological materials are expected to be applied to the fabrication of similar new constructs for applications in various fields other than bone and cartilage regeneration. Finally, it is possible that human ADSCs would behave differently from rat ADSCs under the same experimental conditions adopted in this study; therefore, it is necessary to study larger animals and cells from animal species that are more similar to humans. In addition, the larger the animal, the larger the constructs that need to be fabricated. As the calcification of larger construct is expected to be more difficult from the viewpoint of nutrient supply, improvement of the method of construct preparation and culture for such scale-up remains an important issue for future investigation.
In this study, we fabricated scaffold-free bone- and cartilage-like constructs consisting of ADSCs without AMs. This is the first study to show that scaffold-free ADSCs can maintain their 3D structure and produce the same CT values as the cortical bone without the use of scaffolds or any AMs. Moreover, the chondrogenic-induced constructs are expected to differentiate into bone tissue through endochondral ossification after implantation, and we believe that they can also be applied for cartilage regeneration by ADSCs, similar to our previous findings on cartilage regeneration using iPSCs [41]. As there are no previous reports of scaffold-free bone-like and cartilage-like constructs using ADSCs in vitro, our results highlight the potential of ADSCs for bone and cartilage reconstruction.
RF performed the research and prepared the paper. DM designed and initiated the study, prepared the paper, and interpreted the data. KN conceived and supervised this study. All authors read and approved the final manuscript.
All procedures were evaluated and approved by the Institutional Animal Care and Use Committee of Saga University (application no. A2020-048-0).
The authors thank Editage for language editing support and Advantec Co., Ltd. for help with histology.
This research was funded by the Japanese Society for the Promotion of Science, grant number 19K18503 (to DM).
KN is a co-founder and shareholder of Cyfuse Biomedical K.K. as well as an inventor/developer who holds the patent for the Bio-3D printer. Patent title: Method for production of three-dimensional structure of cell; Patent number: JP4517125. Patent title: Cell structure production device; Patent number; JP 5896104. The other authors declare that there is no conflict of interest regarding the publication of this article.