Objectives. Distraction osteogenesis (DO) mobilises bone regenerative potential and avoids the complications of other treatments such as bone graft. The major disadvantage of DO is the length of time required for bone consolidation. Mesenchymal stem cells (MSCs) have been used to promote bone formation with some good results. Methods. We hereby review the published literature on the use of MSCs in promoting bone consolidation during DO. Results. Studies differed in animal type (mice, rabbit, dog, sheep), bone type (femur, tibia, skull), DO protocols and cell transplantation methods. Conclusion. The majority of studies reported that the transplantation of MSCs enhanced bone consolidation or formation in DO. Many questions relating to animal model, DO protocol and cell transplantation regime remain to be further investigated. Clinical trials are needed to test and confirm these findings from animal studies. Cite this article: Y. Yang, S. Lin, B. Wang, W. Gu, G. Li. Stem cell therapy for enhancement of bone consolidation in distraction osteogenesis: A contemporary review of experimental studies. Bone Joint Res 2017;6:385–390. DOI: 10.1302/2046-3758.66.BJR-2017-0023

Distraction histogenesis (DH) techniques have been widely accepted and practiced in orthopaedics, traumatology, and craniofacial surgery over the last two decades. Using DH methods, many previously untreatable conditions have been successfully managed with outstanding clinical outcomes. The biological mechanisms underlying DH have been studied and the tension-stress principles of tissue regeneration are attributed to upregulated gene expression, enhanced cell proliferation, angiogenesis and tissue remodelling and endogenous stem cell mobilization. The new methods of enhancing bone consolidation in DH are proposed and need further clinical studies. The novel applications of DH have now been extended for the treatment of vascular diseases, cranial defect (with neuronal disorders), hip and spinal deformity corrections and soft-tissue defects in addition to various bone defects and deformities. There are more surprises and novel mechanisms yet to be discovered for these novel applications of DH

Segmental bone transport (SBT) with an external fixator has become a standard method for treatment of large bone defect. However, a long time-application of devices can be very troublesome and complications such as nonunion is sometimes seen at docking site. Although there have been several studies on SBT with large animal models, they were unsuitable for conducting drug application to improve SBT. The purpose of this study was to establish a bone transport model in mice. Six-month-old C57BL/6J mice were divided randomly into bone transport group (group BT) and an immobile control group (group EF). In each group, a 2-mm bone defect was created in the right femur. Group BT was reconstructed by SBT with external fixator (MouseExFix segment transport, RISystem, Switzerland) and group EF was fixed simply with unilateral external fixator (MouseExFix simple). In group BT, a bone segment was transported by 0.2 mm per day. Radiological and histological studies were conducted at 3 and 8 weeks after the surgery. In group BT, radiological data showed regenerative new bone consolidation at 8 weeks after the surgery, whereas high rate of nonunion was observed at the docking site. Histological data showed intramembranous and endochondral ossification. Group EF showed no bone union. In this study, experimental group showed good regenerative new bone formation and was similar ossification pattern to previous large animal models. Thus, the utilization of this bone defect mice model allows to design future studies with standardized mechanical conditions for analyzing mechanisms of bone regeneration induced by SBT

Introduction. Since 2005, we have performed implantation of bone marrow-derived mononuclear cells for osteonecrosis of the femoral head in order to improve vascularization and bone repair. This study focused on early bone repair of osteonecrosis of the femoral head after transplantation of bone marrow-derived mononuclear cells (BMMNC). Patients and Methods. Twenty-two patients (30 joints) who had bilateral osteonecrosis followed for more than 2 years after BMMNC implantation were evaluated. Eight women and 14 men were included. Their mean age at surgery was 41 years (range, 18 to 64 years) and the mean follow-up period was 31 months. Pre-operative stage according to the ARCO classification was Stage 2 in 25 joints and Stage 3 in 5 joints. The mean volume ratio of osteonecrosis was 21%. For preparing BMMNC, about 700ml of bone marrow was aspirated from the ilium and centrifuged using a Spectra cell separator (Gambro). The BMMNC were seeded to interconnected porous calcium hydroxyapatite (IP-CHA) and implanted to the osteonecrotic lesion. As a control, cell-free IP-CHA was implanted for 8 patients (9 joints). A woman and 7 men were included. The mean age at surgery was 49 years (range, 28 to 73 years) and the mean follow-up period was 37 months. Preoperative stage was stage 2 in all patients. The mean volume ratio of osteonecrosis was 22%. At post-operative evaluations; progression of collapse, consolidation at reactive zone, post-operative course of volume rate of osteonecrosis, and bone absorption at osteonecrosis was assessed. Results. Shrinkage of osteonecrosis has been observed subsequent to bone consolidation at the transitional zone which progressed from 3 to 6 months post-operatively in the BMMNC-seeded group. Progression of collapse more than 2 mm was detected in 4 joints (13%), and hip arthroplasty was performed in 1 joint (3%). Consolidation at the reactive zone was detected in 28 joints (93%) and the volume rate of osteonecrosis significantly decreased by 12 months after surgery. Bone resorption at the osteonecrotic lesion was observed in 14 joints (47%). Meanwhile, subtle bone consolidation was detected after 12 months post-operatively in the control group. Progression of collapse was observed in 6 joints (67%) and further surgical treatments were needed in 3 joints (33%). Conclusion. This study found that BMMNC was beneficial to osteonecrosis of the femoral head from the viewpoint of prevention of collapse

Background. Despite the known multifactorial nature of scaphoid wrist fracture non-union, a possible genetic predisposition for the development of this complication remains unknown. This pilot study aimed to address this issue by performing Single Nucleotide Polymorphisms (SNPs) analysis of specific genes known to regulate fracture healing. Materials and Methods. We reviewed 120 patients in a retrospective case-control study from the Hand Surgery Department of Asepeyo Hospital. The case group comprised 60 patients with confirmed scaphoid wrist non-union, diagnosed by Magnetic Resonance Imaging (MRI) and Computed Tomography (CT). The control group comprised 60 patients with scaphoid fracture and complete bone consolidation. Sampling was carried out with a puncture of a finger pad using a sterile, single-use lancet. SNPs were determined by real-time polymerase chain reaction (PCR) using specific, unique probes with the analysis of the melting temperature of hybrids. The X2 test compared genotypes between groups. Multivariate logistic regression analysed the significance of many covariates and the incidence of scaphoid wrist non-union. Results. We found significant differences in subjects who had a smoking habit (p=0.001), high blood pressure (p<0.001), and surgical treatment (p=0.002) in patients with scaphoid non-union. There were more Caucasians (p=0.04) and males (p=0.001) in the case group. Falls were the main mechanism of fracture. The CC genotype in GDF5 (rs143383) was more frequent in patients with scaphoid non-union compared to the controls (p=0.02). CT was prevalent in the controls (p=0.02). T allele in GDF5 was more frequent in patients without non-union (p=0.001). Conclusions. Individuals who were carriers of the CC genotype in GDF5 showed higher susceptibility to suffering scaphoid wrist non-union. Furthermore, being a carrier of CT and T allele suggests that this could be behave as a protection factor against non-union. This is the first clinical study to investigate the potential existence of genetic susceptibility to scaphoid wrist fracture non-union. Level of evidence. Level III, Cross Sectional Study, Epidemiology Study

Background. Delayed bone healing and nonunion are complications of long bone fractures, with prolonged pain and disability. Regenerative therapies employing mesenchymal stromal cells (MSC) and/or bone substitutes are increasingly applied to enhance bone consolidation. Within the REBORNE project, a multi-center orthopaedic clinical trial was focused on the evaluation of efficacy of expanded autologous bone marrow (BM) derived MSC combined with a CaP-biomaterial to enhance bone healing in patients with nonunion of diaphyseal fractures. To complement the clinical and radiological examination of patients, bone turnover markers (BTM) were assayed as potential predictors of bone healing or non-union. Methods. Bone-specific alkaline phosphatase (BAP), C-terminal-propeptide type I-procollagen (PICP), osteocalcin (OC), β-Cross-Laps Collagen (CTX), soluble receptor activator of NFkB (RANKL), osteoprotegerin (OPG) were measured by ELISA assays in blood samples of 22 patients at BM collection and at follow-ups (6, 12 and 24 weeks post-surgery). Results. A significant relationship with age was found only at Visit 6, with an inverse correlation for CTX, RANKL and OC, and positive for OPG. BTM levels were not related to gender. As an effect of local regenerative process, some BTM showed significant changes in comparison to the starting value. In particular, the time course of BAP, PICP and RANKL was different in patients with a successful healing in comparison to patients with negative outcome. The BTM profile indicated remarkable bone formation activity after 12 weeks after surgery. However, the paucity of failed patients in our case series did not allow to prove statistically the role of BTM as predictors of the final outcome. Conclusion. BTM related to bone cell function are useful to measure the efficacy of a regenerative approach based on expanded MSC. Level of evidence. Diagnostic Level IV. Work supported by the EC, Seventh Framework Programme (FP7), through the REBORNE Project, grant agreement no. 241879

Delayed bone healing and nonunion are complications of long bone fractures, with prolonged pain and disability. Regenerative therapies employing mesenchymal stromal cells (MSC) and/or bone substitutes are increasingly applied to enhance bone consolidation. The REBORNE project entailed a multi-center orthopaedic clinical trial focused on the evaluation of efficacy of expanded autologous bone marrow (BM) derived MSC combined with a CaP-biomaterial, to enhance bone healing in patients with nonunion of diaphyseal fractures. To complement the clinical and radiological examination of patients, bone turnover markers (BTM) were assayed as potential predictors of bone healing or non-union. Peripheral blood was collected from patients at fixed time-endpoints, that is at 6,12 and 24 weeks post-surgery for implantation of expanded autologus MSC and bone-like particles. Bone-specific alkaline phosphatase (BAP), C-terminal-propeptide type I-procollagen (PICP), osteocalcin (OC), β-Cross-Laps Collagen (CTX), soluble receptor activator of NFkB (RANKL), osteoprotegerin (OPG) were measured by ELISA assays in blood samples of 22 patients at BM collection and at follow-up visits. A significant relationship with age was found only at 6 months, with an inverse correlation for CTX, RANKL and OC, and positive for OPG. BTM levels were not related to gender. As an effect of local regenerative process, some BTM showed significant changes in comparison to the baseline value. In particular, the time course of BAP, PICP and RANKL was different in patients with a successful healing in comparison to patients with a negative outcome. The BTM profile apparently indicated a remarkable bone formation activity 12 weeks after surgery. However, the paucity of failed patients in our case series did not allow to prove statistically the role of BTM as predictors of the final outcome. Blood markers related to bone cell function are useful to measure the efficacy of a expanded MSC-regenerative approach applied to long bone non-unions. Changes of the markers may provide a support to radiological assessment of bone healing

In a rabbit model we investigated the efficacy of a silk fibroin/hydroxyapatite (SF/HA) composite on the repair of a segmental bone defect. Four types of porous SF/HA composites (SF/HA-1, SF/HA-2, SF/HA-3, SF/HA-4) with different material ratios, pore sizes, porosity and additives were implanted subcutaneously into Sprague-Dawley rats to observe biodegradation. SF/HA-3, which had characteristics more suitable for a bone substitite based on strength and resorption was selected as a scaffold and co-cultured with rabbit bone-marrow stromal cells (BMSCs). A segmental bone defect was created in the rabbit radius. The animals were randomised into group 1 (SF/HA-3 combined with BMSCs implanted into the bone defect), group 2 (SF/HA implanted alone) and group 3 (nothing implanted). They were killed at four, eight and 12 weeks for visual, radiological and histological study.

The bone defects had complete union for group 1 and partial union in group 2, 12 weeks after operation. There was no formation of new bone in group 3. We conclude that SF/HA-3 combined with BMSCs supports bone healing and offers potential as a bone-graft substitute.

We have developed an animal model to examine the formation of heterotopic ossification using standardised muscular damage and implantation of a beta-tricalcium phosphate block into a hip capsulotomy wound in Wistar rats. The aim was to investigate how cells originating from drilled femoral canals and damaged muscles influence the formation of heterotopic bone. The femoral canal was either drilled or left untouched and a tricalcium phosphate block, immersed either in saline or a rhBMP-2 solution, was implanted. These implants were removed at three and 21 days after the operation and examined histologically, histomorphometrically and immunohistochemically.

Bone formation was seen in all implants in rhBMP-2-immersed, whereas in those immersed in saline the process was minimal, irrespective of drilling of the femoral canals. Bone mineralisation was somewhat greater in the absence of drilling with a mean mineralised volume to mean total volume of 18.2% (sd 4.5) versus 12.7% (sd 2.9, p < 0.019), respectively.

Our findings suggest that osteoinductive signalling is an early event in the formation of ectopic bone. If applicable to man the results indicate that careful tissue handling is more important than the prevention of the dissemination of bone cells in order to avoid heterotopic ossification.