By defination from U.S. Food and Drug Administration, bone nonunion refers to that fractured bone is fixed over 9 months old and has not shown radiographic signs of progression toward healing for 3 consecutive months. Bone nonunion means the termination of normal healing process of fracture[6]. X-ray radiographs of bone nonunion show closed bone marrow cavity, sclerosis of broken ends, and pseudarthrosis formation. Once bone defects in limbs reach 10 mm × 6 mm × 6 mm, bone transplantaion is necessary[7]. Refractory bone nonunion greatly influences patients’ daily life and work and is presently one of important promblems during fracture treatment.

Therapeutic methods and existing problems
Electrostimulation, ultrasound wave stimulation, and in vitro seismic waves produce some therapeutic effects on treamtnet of bone nonunion in limbs, but they are still under development and improvement[8-9]. Animal experiments have demonstrated that some moleculars with osteoinductive capacity, such as transforming growth factor-13 family, bone morphogenic protein, or platelet-derived growth factors, could be used to treat bone defects or fracture, but they are mostly under investigation in the clinical application[10-11].
At present, surgery remains a primary and effective method of treating bone nonunion. Refractory bone nonuion in limbs is often accompanied by muscular, tendon, and articular capsule atrophy, osteoporosis, anchylosis, and relatively poor local blood supply. In this study, color Doppler examination prior to transplantaion demonstrated obviously reduced blood flow signal around the bone nonuion in 21 limbs. Broken ends lack of blood supply, which wound lead to secondary bone atrophy and bone defects. The bone grafts, which are used to fill bone defects while bone fixation, would soon substitute host bone if local blood supply is established and local nutrition metabolism is strengthened. Vascularized bone transplantation ensures good blood supply for bone grafts and enhances the survival rate of bone grafts, but it also has drawbacks such as bone absence in dornor area and recurred fracture. Once primary surgery is failured, most patients would not accept a second more troublesome surgery[12]. 

Characteristics of this study and theoretical basis
Autologous bone marrow is the only tissue that contains osteoinductive and confirmative osteogenitor cells, is the first-choice implant materials for treatment of bone nonunion, promotes bone repair, and help speed up the healing of fracture[13-14]. Artificial bone with osteoinductive capacity, natural net-like structure, and certain biomechanical strength can provide a supporting effect for the in-growth of osteocytes[15-16]. Iliac periosteum can be used for treatment of bone nonunion due to the advantages including abundant blood circulation, thick aperture of deep iliac circumflex artery, constant position, easy harvesting, and able to improve local arterial blood supply and promote venous return, which provides basis for reestablishing blood circulation in local bone. In addition, periosteum germinal layer cells can differentiate into osteoblasts, exhibit osteoinductive capacity, better grow towards artificial bone, and finally promote bone healing[17-18]. This is the theoretical basis for treatment of refractory bone nonunion using transplantation of artificial bone with autologous bone marrow combined with iliac periosteum. In the present study, there were two patients in which bilateral ilia had been taken as bone grafts (bone grafts taken from bilateral anterior ilia and unilateral posterior ilia in 1 patient), but vascuralized periosteum was successfully harvested from these two patients, possibly because of large area of ilium periosteum in human body.

Data analysis and significance
The present three methods for treatment of refractory bone nonunion in limbs share similar manipulations and surgical procedures with the exception of different managements of bone nonunion. There was no significant difference in hospitalization time among combination group, bone marrow group, and iliac periosteum groups (P > 0.05). This occurs possibly due to a fact that discharge time is mostly based on the time for sutures removal, which is basically the same.
X-ray photograph score showed that bone callus grew fastest and time for bone healing was shortest in the combination group (P < 0.05). Bone marrow mesenchymal stem cells derived from autologous bone marrow provide unlimited proliferation and bi-directional differentiation and can differentiate into osteoblasts under the condition of induction[19-20]. Artificial bone can provide scaffolds for recipient area to facilitate osteoblast in-growth and induce bone formation, and provide osteoblasts, mesenchymal stem cells, cell stimulating factors for bone nonunion and bone defect regions, can induce other tissue cells in the fractured region to transform into osteoblasts, form new bone tissue, and reach fracture healing[21]. Iliac periosteum transplantation provides enough blood supply for recipient area, which is the key to bone healing. Data from Table 3 show that X-ray score harvested from 1 month after transplantation was higher in the iliac periosteum group than in the combination group and bone marrow group (P < 0.05). This may result from ilium with cortical bone and local pressurization to bone grafts in the broken ends, as demonstrated by early X-ray radiographs.
There were no significant differences in time for bone healing, X-ray scores and limb function scores at 6, 9, and 12 months after transplantation between bone marrow and iliac periosteum groups (P > 0.05). These findings indicate that artificial bone with bone marrow exhibits similar effects on osteogenesis and bone healing to simple iliac periosteum grafts following transplantation into the region of bone nonunion.
The combination group showed the best limb function recovery at 3 months after fixture (P < 0.05). Rapid bone healing facilitates early limb mobilization, and promotes the recovery of limb joint function. During surgery, loosening treatment was performed in some limbs to avoid further muscle and tendon atrophy and joint stiffness.
In the bone marrow group, artificial bone provides basic skeleton structure and the compounded autologous bone marrow induces and promotes bone healing, which is different in approach but equally satisfactory in results and can provide novel thoughts for clinical treatment of bone nonunion aiming at difficult iliac harvesting and non-satisfactory ilium. But artificial bone with autologous bone marrow provides poor blood supply and lacks of nutrition in fracture region, which are the primary causes of long time for bone healing and poor recovery of limb function recovery. Based on this, iliac periosteum grafting was added in the combination group, which provides better local blood supply and is the important guarantee for treating refractory bone nonunion and shortening the time for bone healing.

Therapeutic experience
① During surgery, the shortening of lower limbs was corrected according to design prior to surgery. But if the scar and tendon contracture in lower limbs are severe, limb shortening could not be corrected to protect vessels and nerves from stretch injury. Osteotomy should be performed in the region providing better tissue, followed by external fixation, and limb was extended at 1 mm/d after surgery. ② During surgery, for affected limbs presenting anchylosis, loosening treatment was used concurrently, and limb function exercise (with the aid of CPM machine) should be strengthened following surgery. ③ Accurate vessel anastomosis through the use of microscope is request, and successful primary anastomosis is encouraged. High-quality anastomosis with no injury to vascular pedicle is a guarantee of vascular patency and a key to bone graft survival and bone healing following surgery.