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Engineering hypertrophic cartilage grafts from lipoaspirate for critical‐sized calvarial bone defect reconstruction: An adipose tissue‐based developmental engineering approach

Abstract Developmental engineering of living implants from different cell sources capable of stimulating bone regeneration by recapitulating endochondral ossification (ECO) is a promising strategy for large bone defect reconstruction. However, the clinical translation of these cell‐based approaches is hampered by complex manufacturing procedures, poor cell differentiation potential, and limited predictive in vivo performance. We developed an adipose tissue‐based developmental engineering approach to overcome these hurdles using hypertrophic cartilaginous (HyC) constructs engineered from lipoaspirate to repair large bone defects. The engineered HyC constructs were implanted into 4‐mm calvarial defects in nude rats and compared with decellularized bone matrix (DBM) grafts. The DBM grafts induced neo‐bone formation via the recruitment of host cells, while the HyC pellets supported bone regeneration via ECO, as evidenced by the presence of remaining cartilage analog and human NuMA‐positive cells within the newly formed bone. However, the HyC pellets clearly showed superior regenerative capacity compared with that of the DBM grafts, yielding more new bone formation, higher blood vessel density, and better integration with adjacent native bone. We speculate that this effect arises from vascular endothelial growth factor and bone morphogenetic protein‐2 secretion and mineral deposition in the HyC pellets before implantation, promoting increased vascularization and bone formation upon implantation. The results of this study demonstrate that adipose‐derived HyC constructs can effectively heal large bone defects and present a translatable therapeutic option for bone defect repair.


| INTRODUCTION
The body's repair mechanisms are often unable to spontaneously heal large bone defects resulting from traumas, tumors, infections, and congenital malformations. These defects represent a significant medical concern and socioeconomic burden. Bone is the second most frequently transplanted tissue after blood, with over 2.2 million bone graft procedures performed annually worldwide, representing an economic burden of US $3 billion. 1 Among all clinically available grafts, autologous bone is still considered the gold standard since it combines all necessary properties required for bone regeneration, namely, osteoconduction, osteoinduction, and osteogenesis. 2 However, the current autologous bone grafting procedure can only be used to treat a small number of patients, mainly due to donor site morbidity, limited donor availability, and the high cost and complication rate. 3,4 Several alternatives to autologous bone grafting have emerged, such as allografts, xenografts, and synthetic grafts. These alternatives are available in various forms and in large quantities, but each has specific indications and limitations, and mixed results have been obtained. 5,6 Therefore, there is an urgent need for the development of a new generation of bone substitutes.
Tissue-engineered bone substitutes consisting of cells, biomaterials, and bioactive molecules have become a viable approach for bone defect reconstruction. [7][8][9] The traditional bone tissue engineering (BTE) approach mimics the process of an intramembranous ossification (IMO) pathway, by which mesenchymal stem cells (MSCs) are induced to undergo osteogenic differentiation and subsequently form a bone-like matrix. 10 A major drawback of such strategies is the limited size of the engineered constructs. In vitro osteogenic induction results in extensive matrix deposition on the surface of the construct, which hampers nutrient delivery and makes it difficult to scale up the size. 11 Furthermore, extensive bone matrix on the surface hinders the invasion of blood vessels upon construct implantation. Thus, such strategies often fail due to avascular necrosis and core degradation resulting from poor perfusion. 12,13 Consequently, attention has shifted toward an alternative route of "developmental engineering," which strives to stimulate in vivo developmental processes and initiate natural factors that govern cell differentiation and matrix production. 14,15 In contrast to IMO-based BTE approaches, developmental engineering-based strategies involve engineering cartilaginous constructs by replicating certain aspects of endochondral ossification (ECO). [16][17][18] Briefly, MSCs are induced to differentiate into chondrocytes in vitro to form a hypertrophic cartilage (HyC) construct containing essential "biological instructions" that initiate the ECO process after implantation, and the defect is subsequently repaired via endochondral bone regeneration (EBR). [19][20][21] This strategy offers a solution to the problems associated with limited size and poor vascularization after implantation. Chondrocytes within the cartilage intermediate can intrinsically resist hypoxic environments 22 and induce neovascularization and ossification through the release of bioactive factors, including vascular endothelial growth factor (VEGF), bone morphogenic proteins (BMPs), and hydroxyapatite-containing vesicles. 20,23 Furthermore, engineered HyC grafts have been reported to promote faster host integration and bone formation after implantation in vivo. [24][25][26] However, clinical translation of developmental engineering-based bone regeneration is still in the early stages. One of the main challenges is the complexity and variability of current EBR approaches.
Various types of cells, including adipose-derived stem cells (ASCs), 24,27 bone marrow-derived stem cells (BMSCs), 9,20 induced pluripotent stem cells, 28,29 and periosteum-derived cells (PDCs), 30,31 have been used for bone defect reconstruction via EBR. However, these cell-based strategies frequently rely on a series of in vitro cell manipulation techniques, including cell isolation, in vitro expansion, and seeding onto scaffolds, which not only hamper clinical transplantation but also impair the in vivo performance of the engineered implants. [32][33][34] We previously used fractioned human subcutaneous adipose tissue (nanofat 35 ) rather than ASC-seeded bioscaffolds for endochondral bone engineering. The resulting constructs developed a HyC phenotype and demonstrated better endochondral bone formation capacities than ASC-seeded collagen sponges in ectopic bone formation models. 36 In this approach, adipose tissue not only serves as a stem cell niche for tissue regeneration but also provides an innate extracellular matrix (ECM) that acts as a native scaffold and supports stem cell proliferation and differentiation during matrix synthesis and remodeling. These proof-of-concept studies provide a clinically translatable alternative to bone defect repair; however, preclinical studies in animal models are required to evaluate the feasibility of this approach for repairing bone defects.
In this study, we propose an adipose tissue-based developmental engineering strategy for reconstruction of critical-sized bone defects.
To do so, we engineered HyC constructs from human lipoaspirate by sequential in vitro proliferative culture, chondrogenic differentiation, and hypertrophic induction. The resulting HyC pellets were subsequently implanted into critical-sized calvarial defects in a nude rat model and compared with a bone substitute decellularized and demineralized bone matrix (DBM) graft to evaluate their ability to promote EBR and remodeling in orthotopic sites. SH9H-2021-A974-SB). Human Adiscaf constructs were generated as previously described. 36,37 Briefly, lipoaspirate was washed, minced, and loaded into a 20-ml syringe that was connected to another syringe through a three-way stopcock. Then, the lipoaspirate was fractionated by shifting the content from one syringe to another 30 times. The emulsified adipose tissue, namely, nanofat, 35 was seeded into six-well agarose-coated plates. The nanofat was cultured in proliferative medium for 3 weeks, consisting of alpha-minimal essential medium supplemented with 10% fetal bovine serum, 1% HEPES, 1% sodium pyruvate, 1% penicillin-streptomycin glutamine (all from Gibco), 10 À5 M ascorbic acid, 10 À7 M dexamethasone (both from Sigma-Aldrich), 5 ng/ml fibroblast growth factor-2, and 10 ng/ml platelet-derived growth factor (both from R&D Systems). After 3 weeks, 4-mm biopsy punches were taken from the Adiscaf constructs and placed into 12-well agarose-coated plates for an additional week of proliferative culture.

| Cell isolation, expansion, and differentiation induction
Stromal vascular fraction (SVF) cells harvested from lipoaspirate and ASCs from Adiscaf constructs were used as reference for the efficiency of differentiation capacity. SVF cells were isolated after enzymatic digestion of adipose tissue and centrifugation as previously described. 37 In vitro osteogenic, adipogenic, and chondrogenic differentiation were induced as previously described. 37 Briefly, 5 Â 10 5 cells were seeded onto Ultrafoam (4 mm in diameter, 1-mm thick; Davol). The constructs were then cultured in chondrogenic medium, composed of serum-free CM supplemented with 10 À7 M dexamethasone, 0.01 mM ascorbic acid, 10 ng/ml transforming growth factor-β 3 (TGF-β 3 ), and 10 ng/ml bone morphogenetic protein 6 (BMP-6; both from R&D Systems) for 4 weeks.

| Glycosaminoglycan and DNA quantification
Samples were digested with proteinase K for 16 h at 56 C.

| Scanning electron microscopy
Samples were fixed with 0.25% glutaraldehyde at 4 C overnight and then washed with PBS. The samples were subsequently dehydrated with alcohol, coated with gold, and imaged with an FEI XL-30 SEM microscope (FEI).

| Animal experiments and surgical procedures
For orthotopic implantation, twenty 6-8-week-old male nude rats (Charles) were used to establish the critical-sized calvarial defect model. The rats were anesthetized by isoflurane, the scalp area was shaved, and a midline incision was made to expose the cranium. Two 4-mm diameter defects were drilled by a trephine bur with normal saline irrigation. The bone defects were left untreated (blank group, n = 20), implanted with HyC pellets (HyC group, n = 10) or implanted with DBM (DBM group, n = 10; Shanghai Exceller Biomedical Company) (Figure 4a,b). The periosteum, subcutaneous tissue, and skin were sutured layer-by-layer after implantation. The rats had free access to food and water thereafter. The animals were sacrificed 6 or 12 weeks postimplantation, and the calvarial bones were harvested and examined. For ectopic implantation, DBM grafts were subcutaneously implanted into 4-6-week-old male Balb/c nude mice (Charles), as previously described. 38,39

| Microcomputed tomography
Microcomputed tomography (microCT) data were acquired from the rats immediately and at 6 and 12 weeks postimplantation using highresolution microCT (Skyscan1176) at 50 kV and 400 μA. Transmission images (360 ) were acquired with an incremental step size of 0.25 .

| Histomorphometric quantification
Quantitative analysis of new bone area was performed on representative H&E staining images by ImageJ software (National Institutes of Health). Quantitative analysis of blood vessels was performed on representative Movat's staining images by ImageJ, in which the blood vessels were stained red with luminal structure. Four different fields of view were randomly selected in four specimens within the defect area under Â20 magnification (n = 16). The vessel density was named blood vessels/mm 2 by calculating the number of blood vessels per area of each image. Quantitative analysis of NuMA-positive cells was performed by ImageJ and expressed as NuMA + cells (%). Four different fields of view were randomly selected in four specimens of the HyC group under Â20 magnification (n = 16).

| Statistical analysis
All experiments were performed at least in triplicate per condition.
Data were presented as the mean ± standard deviation. Data were compared with one-way analysis of variance with Tukey's multiple comparison test or Student's t-tests to determine significant differences between two groups. The results were considered significantly different when p values were lower than 0.05. Statistical analysis was performed with GraphPad Prism 9.0 software (GraphPad Software).

| Implantation of HyC pellets significantly accelerated critical-sized calvarial defect healing via ECO
The in vivo performance of the engineered HyC pellets was compared with that of DBM in an orthotopic immunodeficiency rat calvarial model (Figure 5a,b). MicroCT scans were performed immediately and at 6 and 12 weeks postimplantation to investigate bone regeneration and integration. The 3D reconstructive images showed that in the HyC group, newly formed hard tissue could be observed in the defect space and started to integrate into the adjacent native bone at 6 weeks. By 12 weeks, the newly formed bone tissues covered most of the defect space and had extensive integration along the margin of defects (Figure 5c, bottom). In contrast, in the DBM group, little newly formed hard tissue was present within the trabeculae of the implanted DBM at 6 and 12 weeks, and only minimal integration with the surrounding bone could be observed at 12 weeks (Figure 5c, middle). No obvious hard tissue could be observed within the untreated group at the same time points (Figure 5c, above). The coronal view of the microCT images at 12 weeks showed that the newly formed bone tissue in the HyC group integrated well with the adjacent native bone ( Figure 5d). To quantitatively evaluate bone formation in orthotopic sites, bone morphological parameters, including the percentage of BV/TV and BMD, were analyzed. As shown in Figure 5e, when the DBM grafts were calculated as a part of the bone tissue within the defect area, the BV/TV percentages in the HyC and DBM groups were comparable (p = 0.27) and significantly higher (p < 0.001) than those in the untreated group at 6 weeks. After 12 weeks of orthotopic implantation, the BV/TV percentage in the HyC group was markedly increased, approximately 1.6 times higher than that in the DBM group. Meanwhile, the BMD of the HyC group was initially lower (p < 0.001) than that of the DBM group at 6 weeks but gradually increased and higher than that of the DBM group at 12 weeks  (Figure 6a,b, middle). However, no evidence of new bone formation was observed by H&E and Masson staining when the DBM grafts were implanted subcutaneously at ectopic sites in nude mice ( Figure S3a,b). In the HyC group, a substantial level of dense bone tissue was observed in the defect space and integrated well with adjacent native bone at 6 weeks, which had repaired almost the entire  (Figure 6d). Movat's pentachrome staining, by which the cartilage matrix was stained blue, the dense bone tissue was stained red, and the newly formed bone tissue was stained yellow, was performed to investigate the histological characteristics of the newly formed bone tissue within the defects (Figure 6c). In the DBM group, the DBM grafts were stained deep red, and no cells were observed within the grafts. The core of the newly formed bone tissue was stained yellow, and the outer area was stained red, but no blue staining was found in the defect area, indicating that the new bone tissue may have formed via IMO (Figure 6c, middle). In the HyC group, the outer area of the newly formed bone tissue with cuboidal osteoblasts/osteocytes (red arrows) was stained red, and the core consisted of yellow-stained trabecular-like bone matrix and blue-stained cartilage analogs. More importantly, blood vessels (black arrows) and enlarged hypertrophic chondrocytes (blue arrows) were observed in the cartilage analogs, indicating that bone tissue was formed via ECO in the HyC group (Figure 6c, right).

| The newly formed bone tissue originated from human adipose tissue and underwent remodeling in vivo
Hypertrophic chondrocytes have the intrinsic ability to secrete angiogenic factors, which promote capillary invasion and regulate bone regeneration and remodeling during development. 25,45 As shown in shown higher chondrogenic potential differentiation and greater Col X expression capacity than ASCs. 48 ASCs are easily accessible in abundant quantities by a minimally invasive procedure and have a higher proliferative capacity. 11,49 More importantly, ASCs, either cultured as micromass pellets 50,51 or spheroids 45 or seeded onto collagen sponges, 36,50 can mature into HyC tissues under in vitro chondrogenic priming conditions and recapitulate ECO in vivo, suggesting their clinical translation potential for bone defect reconstruction. However, the clinical translation of these ASC-based approaches is still limited by complex cell isolation, expansion, and loading procedures. Furthermore, ASCs easily lose their primitive stemness and exhibit reduced proliferation potential and cell senescence during long-term in vitro expansion. [52][53][54] Evidence that a native ECM microenvironment actively preserves the stemness and differentiation potential of mesenchymal progenitor cells was confirmed in different stem cells, 55,56 including BMSCs, 57 ASCs, 34 and periodontal ligament cells. 58 59 In this study, we confirmed that hypertrophic induction induced abundant VEGF and BMP-2 accumulation and mineral deposition in Adiscaf pellets, which was consistent with the results of previous studies in which the expression of BMPs and VEGF at the mRNA or protein level was also upregulated in HyC constructs engineered from ASCs or BMSCs. 26,45,50 The capacity of HyC pellets to express VEGF and BMP-2 and exhibit mineral deposition may be responsible for the greater percentage of bone volume and higher blood vessel density obtained with these pellets than with the traditional DBM grafts in this study.
We previously reported that HyC constructs engineered from human adipose tissue can remodel into bone organs containing abundant bone matrix and marrow components upon ectopic implantation, indicating their potential for bone defect repair. 36,37 In the present study, we further investigated the capacity of adipose-derived HyC In clinical scenarios, the implanted DBM is usually considered a part of the hard tissue in repaired defects. Even taking this factor into account, the percentage of total BV in the DBM group was comparable to that in the HyC group at 6 weeks but significantly lower at 12 weeks, which may result from the greater new bone formation in the HyC group and possible DBM absorption. In this study, we employed a critical-sized calvarial bone defect model, where was considered to provide an IMO-inducing healing microenvironment, to investigate the bone forming capacity of the adipose tissue-derived HyC pellets. We observed the coexistence of both bone and HyC in the implanted HyC pellets, confirming that the bone formation in the calvarial defect was mainly driven by the HyC pellets themselves. The superior bone regeneration induced by the HyC pellets was likely due to the progression of natural ECO, as was shown for long bone 24,30 or calvarial bone 60 defect repair using chondrocyte pellets implanted into the defects. However, the calvarial bone defect models natively lack mechanical stimuli, which have been proven to promote EBR and neovascular invasion. 61 A major obstacle to clinical translation of this approach is the long-term in vitro endochondral priming period required for preparing HyC pellets before application in the current engineering protocol.
Furthermore, due to immunogenicity and individual variation in the differentiation potential of donor adipose tissue, HyC constructs engineered from human adipose tissue can only be used as autografts, and they mediate bone regeneration in a donor-dependent manner, lending additional uncertainty to clinical translation. One solution for overcoming these issues is developing a decellularized HyC matrix from living HyC pellets as an off-the-shelf and immunecompatible alternative. Recently, decellularized HyC matrices, developed by chemical, enzymatic, and physical procedures 62-65 from different living HyC tissues, have been shown to have the capacity to directly attract endogenous MSCs toward the scaffold by leveraging bioactive cues embedded within the decellularized HyC matrix. 66 These matrices can also be activated by living cells before implantation 67,68 to initiate the ECO process and EBR.

| CONCLUSION
Here, we are the first to demonstrate the feasibility of repairing large bone defects with engineered HyC constructs generated from human subcutaneous adipose tissue, which provide the necessary angiogenic and osteogenic signals following implantation to enhance vascularization, bone formation, and remodeling in vivo. The results demonstrate that the engineered HyC constructs can support bone formation in orthotopic defects by recapitulating the ECO process and showed higher efficiency in promoting bone regeneration, integration, and vascularization than traditional DBM grafts and untreated defects.

CONFLICTS OF INTEREST
The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT
The data that supports the findings of this study are available in the supplementary material of this article.