A novel mouse model to study fracture healing of the proximal femur

The majority of fractures, especially in elderly and osteoporotic patients, occurs in metaphyseal bone. However, only a few experimental models exist to study metaphyseal bone healing in mice. Currently used mouse models of metaphyseal fracture healing are either based on drill hole defects, lacking adequate biomechanical stimulation at the site of fracture and therefore endochondral ossification in the fracture callus, or are introduced into the distal part of the mouse femur stabilized by a locking plate, which is challenging due to the small specimen size. Therefore, the aim of the current study was to develop a new mouse model to study metaphyseal fracture healing of the proximal femur. We chose a combination between an open osteotomy and a closed intramedullary stabilization. A 24 G needle was inserted into the femur in a closed manner, then an osteotomy was made with a 0.4-mm Gigli wire saw between the third and the lesser trochanter of the femur using an open approach. Fractured femurs were analyzed using microcomputed tomography and histology at days 14 and 21 after surgery. No animals were lost due to surgery or anesthesia. All animals displayed normal limb loading and a physiological gait pattern within the first three days after fracture. We found robust endochondral ossification during the fracture healing process with high expression of late chondrocyte and early osteogenic markers at day 14 (d14). By day 21 (d21), all fractures had a bony bridging score of 3 or more, indicating successful healing. Callus volume significantly decreased from d14 to d21, whereas high numbers of osteoclasts appeared at the fracture callus until d21, indicating that callus remodeling had already started at d21. In conclusion, we successfully developed a novel mouse model to study endochondral fracture healing of the proximal femur. This model might be useful for future studies using transgenic animals to unravel molecular mechanisms of osteoporotic metaphyseal fracture healing.     

Comparison of healing process in open osteotomy model and open fracture model: Delayed healing of osteotomies after intramedullary screw fixation

Murine osteotomy and fracture models have become the standard to study molecular mechanisms of bone healing. Because there is little information whether the healing of osteotomies differs from that of fractures, we herein studied in mice the healing of femur osteotomies compared to femur fractures. Twenty CD‐1 mice underwent a standardized open femur osteotomy. Another 20 mice received a standardized open femur fracture. Stabilization was performed by an intramedullary screw. Bone healing was studied by micro‐CT, biomechanical, histomorphometric and protein expression analyses. Osteotomies revealed a significantly lower biomechanical stiffness compared to fractures. Micro‐CT showed a reduced bone/tissue volume within the callus of the osteotomies. Histomorphometric analyses demonstrated also a significantly lower amount of osseous tissue in the callus of osteotomies (26% and 88% after 2 and 5 weeks) compared to fractures (50% and 100%). This was associated with a delayed remodeling. Western blot analyses demonstrated comparable BMP‐2 and BMP‐4 expression, but higher levels of collagen‐2, CYR61 and VEGF after osteotomy. Therefore, we conclude that open femur osteotomies in mice show a markedly delayed healing when stabilized less rigidly with an intramedullary screw. This should be considered when choosing a model for studying the mechanisms of bone healing in mice. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:971–978, 2015.     

Effect of Stabilization on the Healing Process of Femur Fractures in Aged Mice

Background: The influence of mechanical stability on fracture healing has previously been studied in adult mice, but is poorly understood in aged animals. Therefore, we herein studied the effect of stabilization on the healing process of femur fractures in aged mice. Methods: Twenty-four 18-month-old CD-1 mice were stabilized after midshaft fracture of the femur with an intramedullary screw. In another 24 18-month-old mice, the femur fractures were left unstabilized. Bone healing was studied by radiological, biomechanical, histomorphometric, and protein expression analyses. Results: After 2 and 5 weeks of healing, the callus of nonstabilized fractures compared to stabilized fractures was significantly larger, containing a significantly smaller amount of osseous tissue and a higher amount of cartilaginous tissue. This was associated with a significantly lower biomechanical stiffness during the early phase of healing. However, during the late phase of fracture healing both nonstabilized and stabilized fractures showed a biomechanical stiffness of ～40%. Of interest, Western blot analyses of callus tissue demonstrated that the expression of proteins related to angiogenesis, bone formation and remodeling, i.e. VEGF, CYR61, BMP-2, BMP-4, Col-2, Col-10, RANKL, OPG, did not differ between nonstabilized and stabilized fractures. Conclusion: Nonstabilized fractures in aged mice show delayed healing and remodeling. This is not caused by an altered protein expression in the callus but rather by the excessive interfragmentary movements.     

Action of IL‐1β during fracture healing

After bone injury, developmental processes such as endochondral and intramembranous ossification are recapitulated as the skeleton regenerates. In contrast to development, skeletal healing involves inflammation. During the early stages of healing a variety of inflammatory cells infiltrate the injured site, debride the wound, and stimulate the repair process. Little is known about the inflammatory process during bone repair. In this work, we examined the effect of a pro‐inflammatory cytokine, Interleukin‐1 beta (IL‐1β), on osteoblast and stem cell differentiation and on intramembranous and endochondral ossification, because IL‐1β exerts effects on skeletal homeostasis and is upregulated in response to fracture. We determined that IL‐1β stimulated proliferation of osteoblasts and production of mineralized bone matrix, but suppressed proliferation and inhibited differentiation of bone marrow derived MSCs. We next performed loss‐ and gain‐of‐function experiments to determine if altering IL‐1β signaling affects fracture healing. We did not detect any differences in callus, cartilage, and bone matrix production during healing of nonstabilized or stabilized fractures in mice that lacked the IL‐1β receptor compared to wild‐type animals. We observed subtle alterations in the healing process after administering IL‐1β during the early phases of repair. At day 10 after injury, the ratio of cartilage to callus was increased, and by day 14, the proportion of cartilage to total callus and to bone was reduced. These changes could reflect a slight acceleration of endochondral ossification, or direct effects on cartilage and bone formation. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:778–784, 2010     

Small animal bone healing models: standards, tips, and pitfalls results of a consensus meeting

Small animal fracture models have gained increasing interest in fracture healing studies. To achieve standardized and defined study conditions, various variables must be carefully controlled when designing fracture healing experiments in mice or rats. The strain, age and sex of the animals may influence the process of fracture healing. Furthermore, the choice of the fracture fixation technique depends on the questions addressed, whereby intra- and extramedullary implants as well as open and closed surgical approaches may be considered. During the last few years, a variety of different, highly sophisticated implants for fracture fixation in small animals have been developed. Rigid fixation with locking plates or external fixators results in predominantly intramembranous healing in both mice and rats. Locking plates, external fixators, intramedullary screws, the locking nail and the pin-clip device allow different degrees of stability resulting in various amounts of endochondral and intramembranous healing. The use of common pins that do not provide rotational and axial stability during fracture stabilization should be discouraged in the future. Analyses should include at least biomechanical and histological evaluations, even if the focus of the study is directed towards the elucidation of molecular mechanisms of fracture healing using the largely available spectrum of antibodies and gene-targeted animals to study molecular mechanisms of fracture healing. This review discusses distinct requirements for the experimental setups as well as the advantages and pitfalls of the different fixation techniques in rats and mice.     

Stimulation of angiogenesis by cilostazol accelerates fracture healing in mice

Cilostazol, a selective phosphodiesterase‐3 inhibitor, is known to control cyclic adenosine monophosphate (c‐AMP) and to stimulate angiogenesis through upregulation of pro‐angiogenic factors. There is no information, however, whether cilostazol affects fracture healing. We, therefore, studied the effect of cilostazol on callus formation and biomechanics during fracture repair. Bone healing was analyzed in a murine femur fracture stabilized with an intramedullary screw. Radiological, biomechanical, histomorphometric, histochemical, and protein biochemical analyses were performed at 2 and 5 weeks after fracture. Twenty‐five mice received 30 mg/kg body weight cilostazol p.o. daily. Controls (n = 24) received equivalent amounts of vehicle. In cilostazol‐treated animals radiological analysis at 2 weeks showed an improved healing with an accelerated osseous bridging compared to controls. This was associated with a significantly higher amount of bony tissue and a smaller amount of cartilage tissue within the callus. Western blot analysis showed a higher expression of cysteine‐rich protein 61 (CYR61), bone morphogenetic protein (BMP)‐4, and receptor activator of NF‐kappaB ligand (RANKL). At 5 weeks, improved fracture healing after cilostazol treatment was indicated by biomechanical analyses, demonstrating a significant higher bending stiffness compared to controls. Thus, cilostazol improves fracture healing by accelerating both bone formation and callus remodeling. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1880–1887, 2015.     

Use of Huckstep nail in the periimplant femoral shaft fracture

87-year-old female underwent open reduction of distal femoral fracture and internal fixation with locking compression plate and bone graft. She was operated for ipsilateral proximal femoral fractures and stabilized by intramedullary interlocked nail 5 years ago. She developed stress fracture proximal to locked plate. We inserted Huckstep nail after removal of the previous operated proximal femoral nail without removing the remaining plate and screws. At 15 month followup the fractures have united. The Huckstep nail has multiple holes available for screw fixation at any level in such difficult situations.     

Characterization of a closed femur fracture model in mice

OBJECTIVES: The goal of this study was to develop and characterize a closed femur fracture model for mice that can be used for the molecular and genetic analysis of fracture healing. STUDY DESIGN: Longitudinal time study of species-specific fracture healing. METHODS: A protocol was developed for creating reproducible, closed femur fractures in mice. Impending fractures were stabilized by retrograde insertion of a 0.01-inch-diameter, stainless steel wire into the intramedullary canal. The intramedullary wire was held in place with a wedge made from the first 2 mm of a 30-gauge needle. Fractures were produced by 3-point bending. Fracture healing was assessed by radiography, histology, and torsional mechanical testing. RESULTS: The mouse femur fracture technique produced good results with minimal loss of animals. Of the 246 mice used in the study, 22 mice were excluded due to poor fracture quality (8), loss of fracture stabilization (6), or to anesthesia death (8). Radiography showed a consistent pattern of fracture healing between mice with peak fracture callus volume evident at 10 (15 mice) to 14 days (18 mice) after fracture. Fracture bridging was apparent in all 3-week postfracture radiographs (35 mice). Histologic examination of 117 specimens at 9 time points showed chondrocyte differentiation within the fracture callus by 7 days after fracture, endochondral ossification occurring by 10 days after fracture, and bone remodeling evident as early as 3 weeks after fracture. Despite radiologic and histologic evidence of fracture bridging after 3 weeks, torsional mechanical testing of 68 mice at 3, 4, 6, and 12 weeks after fracture (group size of 15 to 18 mice at each time point) indicated that significant increases in structural or material strength did not occur until 6 to 12 weeks after fracture. CONCLUSIONS: Femur fracture healing in mice follows a typical endochondral ossification pathway with fracture bridging occurring approximately 1 week faster in mice than rats. This fracture model is amenable to the molecular and genetic analysis of fracture healing using different inbred, transgenic, and knockout strains of mice.     

4种内固定方法治疗锁骨中段骨折疗效比较

目的比较不同内固定方法治疗锁骨中段骨折的疗效。方法应用重建锁定钢板、锁定解剖钢板、Herbert中空螺钉和交锁髓内钉4种内固定方法治疗111例锁骨中段骨折患者。比较4种手术方法的手术时间、切口长度、术中出血量、骨折愈合时间及肩关节功能。结果患者均获得随访,时间12~18个月。手术时间:重建锁定钢板组长于锁定解剖钢板组、Herbert中空螺钉组和交锁髓内钉组,差异有统计学意义(P0.05)。术中出血量:重建锁定钢板组和锁定解剖钢板组多于Herbert中空螺钉组和交锁髓内钉组,差异有统计学意义(P0.05)。切口长度:重建锁定钢板组和锁定解剖钢板组长于Herbert中空螺钉组和交锁髓内钉组,差异有统计学意义(P0.05)。骨折愈合时间:重建锁定钢板长于锁定解剖钢板组、Herbert中空螺钉组和交锁髓内钉组,差异有统计学意义(P0.05)。4种手术方法肩关节功能JOA评分比较差异无统计学意义(P0.05)。并发症:重建锁定钢板组、锁定解剖钢板组各有2例钢板断裂,Herbert中空螺钉组2例退钉,交锁髓内钉组无并发症发生。结论 4种固定方法治疗锁骨中段骨折均取得满意疗效,Herbert中空螺钉和交锁髓内钉固定治疗锁骨中段骨折较重建锁定钢板和锁定解剖钢板固定更符合生物力学特性,更具合理性。     

A model for intramembranous ossification during fracture healing.

We have developed a method to study the molecular basis of intramembranous fracture healing. Unlike intramedullary rods that permit rotation of the fractured bone segments, our murine model relies on an external fixation device to provide stabilization. In this study we compare stabilized fracture callus tissues with callus tissues from non-stabilized fractures during the inflammatory, soft callus, hard callus, and remodeling stages of healing. Histological analyses indicate that stabilized fractures heal with virtually no evidence of cartilage whereas non-stabilized fractures produce abundant cartilage at the fracture site. Expression patterns of collagen type IIa (colIIa) and osteocalcin (oc) reveal that mesenchymal cells at the fracture site commit to either a chondrogenic or an osteogenic lineage during the earliest stages of healing. The mechanical environment influences this cell fate decision, since mesenchymal cells in a stabilized fracture express oc and fail to express colIIa. Future studies will use this murine model of intramembranous fracture healing to explore, at a molecular level, how the mechanical environment exerts its influence on healing of a fracture.     

肱骨干骨折内固定的选择及应注意的问题

目的正确选择肱骨干骨折手术内固定.方法对107例肱骨干骨折分别进行交锁髓内钉(34例)、加压钢板(29例)、Y型钢板(38例)、螺钉(6例)等内固定,并随访比较分析. 结果所有病例均得到随访,平均随访6.3个月.交锁髓内钉、螺钉内固定组全部愈合;加压钢板内固定组愈合26例,伴桡神经损伤2例;Y型钢板内固定组全部愈合,伴肘关节僵硬2例. 结论根据肱骨干骨折的部位和类型选用适当的内固定方法,骨折愈合良好,可有效的减少并发症的发生.     

Effects of fracture fixation stability on ossification in healing fractures

Temporal distribution of intramembranous and endochondral bone formation was studied in experimental fracture defects in rats under different stability of fracture fixation and fracture environments. Animals were surgically treated with a specially developed external fixation construct: Group 1 had 42 rats with a 0-mm fracture gap with bone ends touching corresponding to an axial stiffness of 265.00 +/- 34.00 N/mm and Group 2 had 42 rats with a 2-mm fracture gap corresponding to an axial stiffness of 30.38+/- 2.07 N/mm. From each group, six animals were sacrificed at 4 days and 1, 2, 3, 4, 6, and 12 weeks. Qualitative histologic and morphometric analyses revealed that less fixation rigidity and increased fracture gap induces a later response of bone formation and greater endochondral bone formation leading to prolonged time for full ossification. Furthermore, in the early phase of fracture healing temporal distribution and histologic characteristics of periosteal and intramedullary bone formation are similar and not influenced by rigidity and fracture environment. Results also showed that if tissues associated with the intramedullary region are preserved, intramedullary bone formation is substantial. Finally, histologic data indicate that woven bone might be a prerequisite for the differentiation process of endochondral bone formation.     

微创经皮锁定钢板与空心螺钉髓内固定治疗锁骨骨折疗效比较

目的比较微创经皮锁定钢板与空心螺钉髓内固定治疗锁骨骨折的临床疗效。方法2011年1月~2012年12月收治锁骨骨折患者76例,分别采用经皮锁定钢板内固定(经皮锁定钢板组,40例)和空心螺钉髓内固定(空心螺钉组,36例)微创手术进行治疗。观察两组术中出血量、手术时间、住院时间、术后并发症发生率以及骨折愈合时间、肩关节功能Neer评分,并进行对比研究。结果两组76例患者均顺利完成手术,无一例发生锁骨劈裂,所有伤口均Ⅰ期愈合。与经皮锁定钢板组比较,空心螺钉组平均手术时间短((35.8±6.6)min VS(56.2±7.4)min),术中出血量少((25.3±9.7)ml VS(43.1±9.5)ml),差异均具有统计学意义(P0.05)。但两组间住院时间及骨折愈合时间比较,差异均无统计学意义(P0.05)。两组术后均无一例发生感染、局部皮肤坏死、骨折不愈合等其他并发症。术后空心螺钉组有3例患者出现轻度旋转改变,予以锁骨带保护3~4周后,骨折顺利愈合。空心螺钉组肩关节功能Neer评分高于经皮锁定钢板组((96.3±3.3)VS(92.6±2.9)),差异具有统计学意义(P0.05)。结论与微创经皮锁定钢板内固定术相比较,空心螺钉髓内固定治疗锁骨骨折具有术中出血少、手术时间短及功能恢复好的优势;只要掌握好其适应证,可获得更好的临床疗效。     

Ex vivo analysis of rotational stiffness of different osteosynthesis techniques in mouse femur fracture

The various molecular mechanisms of cell regeneration and tissue healing can best be studied in mouse models with the availability of a wide range of monoclonal antibodies and gene‐targeted animals. The influence of the mechanical stability of individual stabilization techniques on the molecular mechanisms of fracture healing has not been completely elucidated yet. Although during recent years several osteosynthesis techniques have been introduced in mouse fracture models, no comparative study on fracture stabilization is available yet. We therefore analyzed herein in a standardized ex vivo setup the rotational stiffness of seven different osteosynthesis techniques using osteotomized right cadaver femora of CD‐1 mice. Uninjured femora without osteotomy served as controls. Femur stabilization with a locking plate or an external fixator resulted in a rotational stiffness almost similar to the intact femur. The use of a “pin‐clip” device, a “locking nail,” a “mouse nail,” or an “intramedullary screw” produced a lower torsional stiffness, which, however, was still significantly higher than that achieved with the widely applied conventional pin. By the use of the presented data a more specific choice of stabilization technique will be possible according to the various questions concerning molecular aspects in fracture healing. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 1152–1156, 2009     

Nail or plate fixation for A3 trochanteric hip fractures: A systematic review of randomised controlled trials

Continuing controversy exists for the choice of implant for treating A3 trochanteric hip fractures so we undertook a systematic review of randomised controlled trials from the year 2000 onwards that have compared an intramedullary nail with an extramedullary fixation implant for the treatment of these fractures. Data on the occurrence of any fracture healing complications was extracted and the results combined to calculate Peto odd ratio. Nine studies involving 370 fractures were identified. Three studies involving 105 fractures compared an intramedullary nail with a static fixation (condylar, blade or locking plate). Plate fixation was associated with a fivefold increase risk of fracture healing complications (19/52(36.6%) versus 4/53(7.5%), odds ratio 0.14, 95% Confidence intervals 0.04–0.45). Six studies involving 265 fractures compared an intramedullary nail with a sliding hip screw. No statistically significant difference was found in the occurrence of facture healing complications between implants (13/137(9.5%) versus 11/128(8.6%) odds ratio 0.28, 95% Confidence intervals 0.50–2.80). Bases on the evidence to date from randomised trials, the use of fixed nail plates for surgical fixation of this type of fracture cannot be justified. Intramedullary nail fixation and the sliding hip screw have comparable fracture healing complication rates.     

经皮锁定板微创治疗胫骨远段骨折

目的探讨应用锁定板经皮内固定治疗胫骨远段骨折的疗效。方法采用小切口经皮插入胫骨远段内侧锁定板治疗29例胫骨远段骨折。结果 25例获得随访,时间12-28个月。骨折临床愈合时间12-20周。1例开放性骨折者发生皮肤坏死,换药后愈合。5例切口线结反应,取出线结后愈合。5例骨折延迟愈合（3例为27周,2例33周）,无内固定断裂与松动。按Johnor-Wruhs胫骨骨折临床效果评分法：优15例,良7例,中2例,差1例。结论锁定板经皮微创治疗胫骨远段骨折,创伤小、恢复快、骨折愈合率高,疗效较好。     

Moderate soft tissue trauma delays new bone formation only in the early phase of fracture healing.

The aim of this study was to investigate the effect of a moderate soft tissue trauma to the course of fracture healing in a standardized animal model. Thirty-eight Wistar rats were randomly divided into a fracture group (F, n = 19) and a group with a fracture and a soft tissue trauma (F + STT, n = 19). The fracture and the soft tissue trauma were created using an impact device with a standardized energy. All fractures were stabilized by two Kirschner wires. Three rats were measured for blood flow and sacrificed at days 1, 3, 7, and 14, and seven rats at day 28, from both groups. A three-point bending test was performed on the healed tibia after 28 days. During the first 24 h there was a reduction in blood flow, which was more pronounced in the F + STT group than in the F group. From histological sections, the shape of the callus formation, as well as the tissue distribution of newly formed bone, fibrous cartilage and fibrous connective tissue were determined. Distinctly more periosteal new bone formed and a larger callus formed at days 3 and 7 in group F compared to group F + STT. However, by days 14 and 28, the ossification and overall callus size no longer showed differences between the two groups. A fast recovery of blood flow and callus formation took place in the F + STT group, which led to similar histological and biomechanical results in fracture healing observed after 28 days between the two groups.     

股骨干骨折合并同侧股骨颈骨折治疗的临床观察

目的：探讨股骨干合并同侧股骨颈骨折的治疗特点和不同固定方法的疗效。方法：股骨干骨折合并同侧股骨颈骨折27例,男22例,女5例;年龄14～65岁,平均35岁。动力髋螺钉（DHS）固定3例,加压钢板加空心加压螺钉固定12例,重建钉固定8例,顺行髓内钉加空心加压螺钉固定4例。13例固定术前用克氏针临时固定股骨颈骨折。结果：术后随访36～75个月,平均44个月。25例股骨颈骨折平均愈合时间4.5个月,2例股骨颈骨折不愈合。27例股骨干均愈合,平均愈合时间6个月。未用克氏针临时固定股骨颈骨折14例中,2例出现股骨颈不愈合,3例轻度髋内翻畸形。结论：股骨干合并同侧股骨颈骨折有许多固定方法可供选择,加压钢板加空心加压螺钉固定简便易用,在实施固定术前用克氏针临时固定股骨颈骨折可避免股骨颈骨折再移位和损伤。     

Therapeutic possibilities in trochanteric fractures. Safe--fast--stable

Trochanteric fractures frequently occur in elder patients with severe osteopenia. These fractures are highly unstable. Conservative treatment is not indicated because of the long period of immobilization until fracture healing is achieved. Simple fractures (A1) can be successfully fixed by either plate-hip screw systems like Dynamic Hip Screw (DHS) or nail-hip screw systems (Gamma-Nail, Proximal Femur Nail, Classic Nail). As a result of biomechanical and clinical trials the intramedullary implants should be preferred for fixation of more comminuted fractures (Type A2.2, A3.3) and subtrochanteric fractures. For ipsilateral femoral shaft and trochanteric fractures long nail-hip screw-systems are recommended like the long PFN or the long Gamma nail. In about 90% of the patients with trochanteric fractures postoperative weight bearing can be achieved by internal fixation if gliding systems are applied. Intraoperative and postoperative complications however occur more frequently after the implantation of Gamma-Nail than after application of a DHS. Other implants like Ender Nails, 130 degrees angled plate or twisted plate are not further recommended.     

Comparison of Fracture Healing Among Different Inbred Mouse Strains

Quantitative trait locus analysis can be used to identify genes critically involved in biological processes. No such analysis has been applied to identifying genes that control bone fracture healing. To determine the feasibility of such an approach, healing of femur fractures was measured between C57BL/6, DBA/2, and C3H inbred strains of mice. Healing was assessed by radiography and histology and measured by histomorphometry and biomechanical testing. In all strains, radiographic bridging of the fracture was apparent after 3 weeks of healing. Histology showed that healing occurred through endochondral ossification in all strains. Histomorphometric measurements found more bone in the C57BL/6 fracture calluses 7 and 10 days after fracture. In contrast, more cartilage was present after 7 days in the C3H callus, which rapidly declined to levels less than those of C57BL/6 or DBA/2 mice by 14 days after fracture. An endochondral ossification index was calculated by multiplying the callus percent cartilage and bone areas as a measure of endochondral ossification. At 7 and 10 days after fracture, this value was higher in C57BL/6 mice. Using torsional mechanical testing, normalized structural and material properties of the C57BL/6 healing femurs were higher than values from the DBA/2 or C3H mice 4 weeks after fracture. The data indicate that fracture healing proceeds more rapidly in C57BL/6 mice and demonstrate that genetic variability significantly contributes to the process of bone regeneration. Large enough differences exist between C57BL/6 and DBA/2 or C3H mice to permit a quantitative trait locus analysis to identify genes controlling bone regeneration.