Non-union remains a challenging problem for orthopaedic surgeons, as it delays recovery and often requires multiple follow-up procedures. Numerous methods of treatment, such as autologous bone graft and allograft bone chips, are available for this condition; however, they are often associated with drawbacks and high costs.23 In recent years, mesenchymal stem cell implantation has garnered interest worldwide due to its regenerative capacity.
Moreover, both preclinical and clinical studies have shown that MSC can aid in bone regeneration.15,24−27 The most common source of MSC is bone marrow; however, its isolation requires an invasive procedure, often causing pain and discomfort to the donors. Other accessible sources include adipose and umbilical cord tissue, which both obviate the need for invasive isolation as they are byproducts of liposuction and childbirth. In this study, we investigate the safety and efficacy of ADSCs and UC-MSCs for treating nonunion.
In this study, 5 (62.5%) subjects developed union in 2–12 months. To our knowledge, this is the most extensive series of allogeneic MSCs in subjects with nonunion. Previously, we administered allogeneic UC-MSCs for infected non-union femoral shaft fracture with a 12 cm bone defect.15 There was a reduction of VAS, from a median of 1 (0–6) to 0 (0–4), and increase in mean LEFS/DASH of 56.25 ± 10.71 to 65 ± 22.72 in one year of follow-up. In an animal model, Qu et al. found that the administration of UC-MSCs resulted in disappearance of fracture line at eight weeks.28 Most subjects still have significant LLD following the procedure that may interfere with daily activities. However, the primary outcome of this study is to achieve union, while this issue can be managed with additional procedures following bone union, such as bone lengthening using the Ilizarov apparatus or lengthening and intramedullary nailing. Regardless the rarity of the occurrence, it should be noted that this procedure might expose the subjects to risks of complications, such as pain, infection, and failure of treatment.29−31
The mechanisms by which exogenous MSC implantation enhances fracture healing have been extensively studied. It was previously postulated that fracture healing occurs due to differentiation of transplanted cells; however, in non-unions without critical bone loss, the implanted MSCs largely act as cellular modulators, instead of directly differentiating, as the differentiation of transplanted MSC at the site was less efficient.15,32−34 Contrary to previously held beliefs, it is the release by MSCs of their secretome, an assemblage of paracrine factors (e.g. cytokines, growth factors, etc.), into the extracellular environment that plays the crucial role in fracture healing.34,35 Several cytokines contained in secretome include insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), and hepatocyte growth factor (HGF). These factors enhance migration and recruitment of osteoprogenitor cells (osteoblasts) to the implantation site and assists in upregulation of cell proliferation and differentiation.15,35 Besides, these factors also aid in angiogenesis, in order to fulfill oxygen and nutritional requirements during fracture healing.35
In comparison from other types of stem cells, MSC poses a shallow risk when implanted. Implantation of exogenous MSC, whether differentiated or undifferentiated, does not cause alloreactive lymphocyte proliferation and does not elicit further immune responses. This suggests that MSC is a safe and potential alternative therapy for treating nonunion.
Contact between fracture fragments should be established to assist bridging between the two fragments. In several cases in our study, we utilized double-plating to ensure contact, alignment, and mechanical stability of the fracture site. In a report by Steinberg et al., double-plating may help in aiding mechanical stability in femoral distal or supracondylar fractures by reducing lever arm and load on the fracture size, which improves fracture stabilization and prevents implant loosening.36,37 We also performed dynamization on subject Bub (case no. 4) by removing the distal screw in the tibia after intramedullary (IM) nailing. Dynamization may promote fracture healing and can be proposed as the primary treatment in non-union cases.6,38,39 However, it should be taken into consideration that the use of intramedullary reaming and nailing, in particular, affects the microvasculature of the bone. As demonstrated in an animal study, bone reaming may destroy the endosteum microvasculature, which is usually followed with vasculature hypertrophy of the periosteum.40
Status of infection may affect bone healing. Changes in the biological environment caused by open fractures may alter the healing process and lead to further septic complications.41 Infection, in coexistence with metal fixtures, may cause osteolysis, loosening, and mechanical failure, eventually making union harder to achieve.42,43 Therefore, eradication of infection must be assured to establish bone union. Radical resection should be performed as needed, debriding all infected and potentially infected non-viable tissue.44 One approach to aid eradication of infection is by performing the Masquelet procedure, which includes local antibiotic delivery as its first step. Subject compliance is another external aspect to be taken into consideration, as antibiotic therapy may be prolonged in this condition.
We observed several complications during our study, in which three experienced infections during follow up. Tissue culture examination revealed two methicillin-resistant Staphylococcus aureus infection. Both subjects were referred to the tropical diseases and infection specialists in our hospital and treated with appropriate antibiotics according to the sensitivity test. Subject 7 (Tas) was administered oral rifampicin and trimethoprim/sulfamethoxazole and underwent sequestrectomy, debridement, and antibiotic-loaded bone cement application. Subject MYu (case no. 2) was given oral trimethoprim/sulfamethoxazole. Multiple MSC implantations with Masquelet procedure stage II were performed on Subject 3 (Suy); however, radiological union has been achieved. The subject complained of mild to moderate pain during follow up (VAS: 3–5) and wound dehiscence. Culture examination revealed Escherichia coli infection, and the subject was treated with oral trimethoprim/sulfamethoxazole according to the sensitivity test. The subject was planned for another debridement and another round of the Masquelet procedure.
Currently, the application of bone morphogenetic protein (BMP) as an osteoinductive agent in treatment is being investigated. It is hypothesized that the addition of recombinant human bone morphogenetic protein 2 (rhBMP2) may assist proliferation and differentiation of MSC, while recombinant human bone morphogenetic protein 7 (rhBMP7) may support osteoblast differentiation.45 Unfortunately, results of multiple studies regarding the use of BMP remain inconclusive.46,47 We are further exploring the possibility of incorporating BMP and MSC treatment.