Diabetes mellitus (DM) is a chronic metabolic disease characterized by the presence of elevated blood glucose levels, and is of two types: type 1 diabetes, which is due to severe deficiency in insulin synthesis, and type 2 diabetes which is caused by insulin resistance combined with insulin production deficiency (Antar et al., 2023). DM is a systematic disease affecting not only the heart, brain, kidneys, eyes, blood vessels and nerves, but it also causes a negative effect on the skeletal system. It is well known that DM contributes to osteoporosis, increased fracture risk, delayed bone healing and impaired bone regeneration (Antar et al., 2023; Csonka and Lendvay, 2023). In fact, it was reported that diabetic patients had a 6- to 7-fold increased risk of hip fracture, 2.75-fold increased risk of implant failure and 1.6-fold delay in fracture healing compared with non-diabetic population (Hu et al., 2018; Hygum et al., 2019). With the rise in incidence of DM and aging of population, the number of DM patients requiring bone reconstruction procedures rapidly increases. However, the repair of bone fracture and defect in diabetic patients still remains a major clinical challenge (Hu et al., 2018). Thus, it is essential to develop effective methods to accelerate bone regeneration in DM patients.

Bone tissue engineering has recently emerged as a promising treatment strategy for the repair and functional regeneration of large segmental bone defects (Li et al., 2018). Its general principle involves the integration of seed cells, tridimensional biomaterial framework and molecular signals to generate an implantable construct (Perez et al., 2018). This approach has been proved effective in both animal models and clinical trials (Confalonieri et al., 2018; Mazzoni et al., 2023). Nevertheless, most of the studies were performed in healthy recipients with unimpaired bone regeneration activity. There are limited studies on whether bone tissue engineering approach could improve bone regeneration under diabetic condition.

One of the fundamental components of bone tissue engineering strategy is seed cells. Bone marrow-derived mesenchymal stem cells (BMSCs) are a major source of osteoblasts, osteocytes and bone lining cells in vivo, and are crucial for bone remodeling via both cellular and paracrine effects. Because of their easy acquisition, potent proliferative and osteogenic potential, well-defined osteogenic differentiation pathway and low immunogenicity, BMSCs are considered as an attractive cellular source for bone tissue engineering applications (Zhang et al., 2012; Grelewski et al., 2023). Although autologous cell sources are generally preferred in clinical trials, it is well documented that BMSCs derived from diabetic patients exhibited decreased proliferative and osteogenic potential (Fijany et al., 2019). In addition, it was reported that diabetic donors possessed fewer BMSCs and that these cells exhibited impaired proliferation and survival in vitro (De Vyver, 2017). On the other hand, the use of allogenic BMSCs derived from healthy donors with efficacious osteogenic potential may circumvent the limitations of autologous BMSCs. Besides, allogenic BMSCs can be easily cultured, expanded and cryopreserved, allowing off-the-shelf availability. Investigations into their use in healing critical-sized bone defects in various animal models demonstrated their utility for bone tissue engineering applications (Berner et al., 2013; Arthur and Gronthos, 2020).

Decalcified bone matrix (DBM) is an artificial bone material obtained by decalcifying biological bone. It is considered as an ideal bone regeneration scaffold due to its good biocompatibility and osteogenic activity (Liu et al., 2023). Previously, we have conducted experiments to investigate the potential of allogenic fetal BMSC-based bone tissue engineering strategy for healing critical-sized bone defect in a rabbit model of osteoporosis (Wang et al., 2015). In the current study, we seeded allogenic fetal BMSCs on DBM scaffolds to construct tissue-engineered bone grafts, and investigated their potential to form both ectopic and orthotopic bone in streptozotocin (STZ)-induced type 1 diabetic rat model.

Diabetes is a group of metabolic disorders which can cause multiple organ damages. Patients with diabetes are more prone to osteoporosis, bone fracture and impaired bone healing than the non-diabetic population (Hofbauer et al., 2022). The rapidly expanding DM patients requiring bone reconstruction procedures motivated the present study to investigate the potential of bone tissue engineering approach to improve bone regeneration under diabetic condition. We implanted BMSC/DBM constructs or cell-free DBM scaffolds in both ectopic and orthotopic bone formation rat models. Our data suggested that BMSC/DBM constructs could form ectopic and orthotopic bone under diabetic condition, but with impaired bone regeneration activity.

It is well known that BMSCs derived from diabetic patients showed decreased alkaline phosphatase (ALP) activity, mineralization and osteogenic gene expression (Silva et al., 2015). It is also proposed that the balance of BMSC differentiation is switched to favor adipogenic differentiation under diabetic condition (Fijany et al., 2019). Besides, a number of studies suggested that the outcomes of autologous MSC therapy for diabetes-associated complications were variable and not as effective as initially hoped, due to the intrinsic MSC dysfunction (De Vyver, 2017). Therefore, autologous BMSCs may not be the ideal candidate to induce and improve bone regeneration under diabetic condition. Allogenic BMSCs have been shown to be nonimmunogenic in vivo transplantation paradigms (Squillaro et al., 2016), and have been used to repair critical-sized bone defects in various animal models (Lin et al., 2019), suggesting their utility for bone tissue engineering applications. In our study, no signs of serious adverse immune reactions were observed. Compared with perinatal and adult sources of MSCs, fetal BMSCs exhibited greater cell proliferative capability, osteogenic potential and lower immunogenicity as reported by previous studies (Zhang et al., 2009; Guillot et al., 2008), thus we chose allogenic fetal BMSCs as our seed cells in the present study.

Our data indicated that BMSC/DBM constructs could form ectopic bone under diabetic condition, but with decreased bone regeneration activity. Previous studies suggested that the osteogenic cells that formed new bone in subcutaneous ectopic bone formation models were mainly of donor origin rather than originated from the local microenvironment (Abdallah et al., 2008; Mankani et al., 2008). Deng et al. demonstrated that BMSCs derived from healthy donors treated with diabetic serum showed a remarkable decrease in osteogenic activity, suggesting that pathologic conditions like diabetes could alter the function of BMSCs derived from healthy donors (Deng et al., 2018). There have been a large number of studies trying to clarify the precise mechanism underlying the detrimental effect of diabetes on BMSCs. It is proved that many factors from the diabetic microenvironment could contribute to the dysfunction of BMSCs, including hyperglycemia, chronic inflammation and increased advanced glycation end-products (AGE) formation (Kume et al., 2005; Li et al., 2007; Yang et al., 2010; Wang et al., 2013; Ko et al., 2015; Qu et al., 2018; Xu and Zuo, 2021). We suggested that the allogenic BMSCs introduced through bone tissue engineering approach were affected by various factors from the diabetic host environment, resulting in the impaired bone regeneration activity. It is worthwhile to track and examine the function of the implanted BMSCs in the future.

Apart from testing the pro-bone regeneration potential of tissue-engineered bone constructs in subcutaneous ectopic bone formation models, we also investigated their potential to repair large segmental bone defects in rat femurs. To our surprise, at 6 months after surgery, BMSC/DBM constructs under diabetic condition showed comparable bone volume and density compared with the non-diabetic controls. However, X-ray analysis at the 1st and 3rd month post-surgery indicated that the healing process was delayed in the diabetic group. The implantation of BMSC-seeded DBM scaffolds enhanced bone formation in orthotopic defects compared with the implantation of unseeded DBM scaffolds, suggesting that the transplanted allogenic BMSCs were able to induce and promote osseous regeneration in orthotopic defect models. However, the delayed healing process compared with non-diabetic controls indicated that the implanted BMSCs were affected by the diabetic host environment, resulting in impaired osteogenic activity. Unlike ectopic bone formation models, previous studies have shown that new bone formation in orthotopic defect models is induced by both host and donor cells, and implantation of donor MSCs can cause recruitment of the host cells (Zhou et al., 2015; Westhauser et al., 2017; Kim et al., 2020). We assume that the implanted BMSCs were able to recruit endogenous stem/progenitor cells to the defect site, and these recruited local stem/progenitor cells together with the transplanted allogenic seed cells were able to induce bone regeneration, eventually leading to bone bridging at the defect site. Future studies could define the origin of the cells that form new bone by labeling the seed cells with fluorescent marker and investigate the interaction between the host and donor cells. The difference in the outcomes of ectopic bone formation model and orthotopic defect model in our study may be attributed to different local environment (ectopic and orthotopic) as well as different osteogenic process (calcification and new bone formation).

Strategies to promote bone regeneration include providing stem/progenitor cells, growth factors, nutrients, and synthetic materials to the defect site to induce tissue regeneration (Shui-Ling et al., 2018). The concept of bone tissue engineering involves the combination of stem/progenitor cells, tridimensional biomaterial scaffolds and growth factors. Although bone tissue engineering approach seems to hold great potential in improving bone regeneration under diabetic condition, the implanted exogenous seed cells would inevitably be affected by the host diabetic microenvironment. Previous studies on bone regeneration strategy under diabetic condition mainly focused on osteoblasts or osteoclasts by delivering pro-osteogenic factors (such as BMP2, VEGFA, FGF-9, IGF-1, Vitamin D, etc.) (Wang et al., 2010; Wu et al., 2013; De Santana and Trackman, 2015; Wallner et al., 2015) or anti-bone resorption factors like parathyroid hormone-related protein (Ardura et al., 2016). A recent study by Hu et al. highlighted the importance of modulating the diabetic microenvironment. They incorporated interleukin 4 into the nanofibrous heparin-modified gelatin microsphere to polarize proinflammatory M1 macrophages into an anti-inflammatory M2 phenotype that facilitate osteogenic differentiation and bone formation (Hu et al., 2018). Their research demonstrated that modulating the diabetic microenvironment played a crucial role in improving bone regeneration under diabetic condition. Future studies could devote to combining these pro-bone regeneration strategies like modulating the diabetic microenvironment with bone tissue engineering approach to accelerate bone regeneration under diabetic condition.

In the present study, we demonstrated tissue-engineered bone constructs could form both ectopic and orthotopic bone in diabetic rats with impaired bone regeneration activity. Our research suggest that bone tissue engineering approach provides a promising way to accelerate bone regeneration under diabetic condition. In future studies, tissue-engineered cell/scaffold constructs could be combined with pro-osteogenic and immunomodulating factors to accelerate bone regeneration under diabetic condition.