A Model for Tissue Engineering Applications: Femoral Critical Size Defect in Immunodeficient Mice

Abstract

Animal models for preclinical functionality assays lie midway between in vitro systems such as cell culture and actual clinical trials. We have developed a novel external fixation device for femoral critical size defect (CSD) in the femurs of immunodeficient mice as an experimental model for studying bone regeneration and bone tissue engineering. The external fixation device comprises four pointed rods and dental acrylic paste. A segmental bone defect (2 mm) was created in the midshaft of the mouse femur. The CSD in the femur of the mice were either left untreated or treated with a bone allograft, a cell-scaffold construct, or a scaffold-only construct. The repair and healing processes of the CSD were monitored by digital x-ray radiography, microcomputed tomography, and histology. Repair of the femoral CSD was achieved with the bone allografts, and partial repair of the femoral CSD was achieved with the cell scaffold and the scaffold-only constructs. No repair of the nongrafted femoral CSD was observed. Our results establish the feasibility of this new mouse femoral model for CSD repair of segmental bone using a simple stabilized external fixation device. The model should prove especially useful for in vivo preclinical proof-of-concept studies that involve cell therapy-based technologies for bone tissue engineering applications in humans.

Introduction

The successful repair of large bone defects that result from either trauma or tumor resection is one of the major goals of tissue engineering. Stimulations resulting from load-bearing situations in bones are critical for promoting osteogenesis, because mechanical stimuli are known to modulate the morphogenesis and regeneration of skeletal tissues. Hence, the search for an ideal method and/or biomaterial that can promote osteogenesis and bone regeneration is crucial for the successful repair of a large bone defect.

An optimal biomaterial should combine the essential characteristics of bone, namely osteoinductivity and osteoconductivity. Moreover, the scaffolds should be biocompatible, biodegradable, and have similar mechanical properties to that of bone.^1–5 Scaffolds for bone repair have been fabricated using a wide range of biomaterials, such as synthetic polymers, native polymers (hydrogels), and their composites.^6–9 Since the inorganic component of natural bone is composed of hydroxyapatite, ceramics such as calcium phosphate, calcium sulfate, and calcium carbonate (coral-based ceramics) have also been used as matrices for bone regeneration.^3,10

Tissue engineering protocols that include integration of osteoprogenitor cells within scaffolds are a promising strategy for bone repair,^11–16 and in recent years, considerable effort has been devoted to developing scaffolds that are impregnated with bone marrow-derived stromal cells (BMSCs). The results of both in vitro^17–19 and in vivo^20–23 mechanical tests on BMSC-impregnated scaffolds have shown that pressure, strain and fluid flow can influence the osteogenic differentiation of BMSCs in vitro as well as on bone regeneration in vivo. In addition, a perfusion culture of BMSCs showed more osteoblastic differentiation than that of static cultured constructs.^{17–19,24,25}

With the development of cell-impregnated scaffolds, small immunodeficient experimental animals, such as athymic nude rats, have been found to be very suitable for studying the potential clinical utility and functionality of scaffolds that are impregnated with human cells, because they do not require immunosuppressive medication. Further, these animals enable xenogeneic cell transplantation without the risk of graft rejection.^26,27

Preclinical bone tissue engineering assays are currently the only method for evaluating the functionality of cell-impregnated scaffolds and other bone substitutes that are intended for human use. The standard method for evaluating bone substitutes is creating a critical size defect (CSD) in the long bones of large experimental animals, such as sheep, to simulate the load-bearing condition. More recently, the laboratory rat with a CSD in the femur or tibia has replaced these large animals for evaluating bone substitutes.²⁸ However, models of orthotopic CSDs in laboratory rats are lacking standardization, and the size of CSD in the long bones of rats in most studies ranges between 2 and 10 mm.²⁹

Genetically engineered mice are currently being used to investigate human bone and skeletal disorders, and it is necessary that a valid and reproducible CSD model will be applied to mouse femur or tibia to cope with bone-tissue-related questions that require in vivo testing using a CSD.

Several reports have recently been published on fixation devices after CSD creation for use in mice and other small rodents. Femoral fixation after CSD creation can be done by either external or internal fixation. For external fixation, a polyethylene rod, for example, is attached to the fractured bone. For internal fixation, an orthopedic implant that is made from biocompatible stainless steels or synthetic materials is placed in the femoral shaft and fixed by insertion into the supracondylar metaphysic of the femur. Both fixation methods have advantages and disadvantages.^26,30–38 Hence, most studies that assessed the functionality of cell-impregnated scaffolds and other bone substitute materials have been done on ectopic and cranial defect models in immunodeficient mice.³⁹ However, these defect models are unsuitable for studying the potential clinical utility of cell-impregnated scaffolds and other bone substitute materials for repairing load-bearing long bones, the primary target for future clinical applications.

Therefore, the aim of this study was to develop a mouse model in which a load-bearing long bone could be used for assessing the potential clinical utility of a cell-impregnated scaffold. This report describes the creation of a 2 mm CSD in the femur of immunodeficient mice and a method of external fixation, including the evaluation of the CSD, the bone allografts, and the cell-scaffold transplants in this mouse model. Since the usual size of a CSD in rat long bones is 6 mm²⁹ and the length of the long bones of the rat is about thrice longer than those of the mouse, a 2 mm size CSD was created in this mouse model.

Materials and Methods

Surgical procedure

The design of an external fixation frame for the mouse femur with a CSD was based on that described by Ben-Ari et al. for repairing a rat's fractured tibia.⁴⁰ The method has been modified for application on segmental bone defect on rat tibiae and adapted for use in the mouse femur. The surgical protocols were approved according to institutional guidelines of the Animal Ethics Committee of the Technion–Israel Institute of Technology, Haifa, Israel. Athymic Nude-Foxn1^nu mice, 8-week-old and weighing 25 g (Harlan Laboratories, Jerusalem, Israel) were used for the experiments.

The mice were anesthetized with a 0.5 mL intraperitoneal injection of a 1:1 mixture of xylazine and ketamine. Under aseptic conditions, a longitudinal incision was made over the lateral aspect of the thigh, which was raised and fixed with silicon (Zetaplus, Shermack, Italy). Muscles were split at the linea alba to expose the femur proximally and distally to the medial aspect of the femoral condyle. Four holes (0.3 mm diameter), two holes in the distal region and two holes in the proximal region, were drilled in the midshaft of the femurs. Rods (27G, 0.4 mm) were inserted manually into the drilled holes by penetration through one lateral cortex to the opposite cortex and through the skin. The protruding ends of the rods on the lateral and medial sides were first bent and then connected with acrylic dental paste (Unifast Trad, GC America, Inc., Alsip, IL). Under saline irrigation, a bone defect of 2 mm between the rods was created in the cylindrical midshaft part of the femur (total length 8 mm) using a motorized mini-drill. The muscles were opposed over the bone defect, and the wound was closed with vicryl sutures (Fig. 1).

FIG. 1.

(A) The appearance of the mouse hind leg after surgery. (B) An x-ray image taken on the day of surgery showing the induced defect with annotations. Color images available online at www.liebertonline.com/tec.

The external fixation frame weighed between 0.6 and 0.8 g. Approximately 1 h after surgery, the mice regained consciousness and started to move in the cage after a few hours. The mice were housed separately in plastic cages. All animals recovered well from the surgery with no visible restrictions in their movements. Animals were weighed before and at the end of the experiment, and no body weight loss was observed as a result of the surgery. Food and water were available ad libitum.

Mechanical testing

To analyze the mechanical properties of the externally fixated bone, the bending stiffness of mice bones was studied. A fixated femur was clamped at the femoral head. Vertical forces that ranged from 5 to 35 g were applied to the foot. The respective deflections of the femurs were measured with dial micrometers (1 mm accuracy) that were placed in front of and behind the fixators. The test was made with four different fixated or intact femurs. Detailed data regarding the mechanical testing are available in Supplementary Material (Supplementary Material and Data are available online at www.liebertonline.com/tec).

CSD and bone allograft

The external fixation device provided a stable frame to tightly hold the bone before creating the CSD. Eight animals were used for each of the four groups (nongrafted CSD, bone allograft group, cell-scaffold group, and scaffold-only group).

To determine which defect size was critical in the femoral midshaft of mice, preliminary tests were conducted on creating a segmental defect of the following sizes: 2.0; 2.5; 3.0 mm. It was found that with defects of 2.0 mm the defect was maintained throughout the 8-week period. The larger CSD (2.5 mm and 3.0 mm) were found to interfere with the stability of the external fixation device (Supplementary Fig. S6). Consequently, a 2.0 mm gap was chosen as the optimal size of the CSD in this assay.

Bone allografts were obtained from bones of littermates of the experimental mice. The allografts were prepared by carefully removing all the attached muscles from the bones. The allograft was cut from the cleaned bones using a rotator drill and then fitted into femoral CSD.

Cell source and culture conditions

BMSCs were isolated from fresh human bone marrow of two male and two female Afro-American donors, aged 21–44 (Cambrex-Lonza Walkersville, Inc., Walkersville, MD) based on their”. The progenitor cells were cultured (37°C, 5% CO₂, 1 week) in a standard culture medium (αMEM) that was supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Biological Industries, Beit Haemek, Israel). At 70–80% confluence, the cells were trypsinized, counted, and passaged (P₁) to induce osteogenic differentiation in culture; the P₁ cultures were cultured in αMEM induction medium that contained 10% FCS, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 100 μg/mL ascorbic acid, and 10⁻⁸M dexamethasone until confluence was achieved about 1 week later. The osteogenic potential of the cells in vitro is shown in Supplementary Fig. S5.

In vivo transplant, in vivo cell-scaffold transplant, and scaffold-only transplant

Confluent cultures (P₂) were trypsinized, washed with phosphate buffered saline (PBS), and 1×10⁶ cells were mixed with ProOsteon particles (Interpore Cross, Irvine, CA; particle size 0.5–1.0 mm, 20 μL volume). These calcium carbonate particles, which were covered with a 5 nm layer of hydroxyapatite (coral/hydroxyapatite scaffold), were rotated gently in an incubator at 37°C for 1 h. At the end of the incubation, the particles were collected by brief centrifugation. The particles were first mixed with mouse fibrinogen (15 μL; 3.2 mg/mL in PBS) and then mixed with mouse thrombin (15 μL; 25 U/mL in 2% CaCl₂) to create fibrin clots. The fibrin clots were then transplanted into the CSD for 8 weeks. Control animals were similarly transplanted with scaffold only.

Postsurgery analysis

Digital x-ray, microcomputed tomography

Digital radiographs of mice femurs were taken on the day of the surgery, and then every 2 weeks for 8 weeks using an Oralix AC Densomat X-ray machine (Gendex Dental System, Milan, Italy) at the following operating conditions: 65 kV peak voltage, 7.5 mA anode current, and an exposure time of 0.26 s) that was operated by KODAK Dental Imaging Software version 6.5 (Kodak Dental Systems, GA).

After 8 weeks, the mice were euthanized and transplanted femurs were dissected, fixed in 10% neutral buffered formalin, and the metal rods of the fixation device were carefully removed before the bones were analyzed by microcomputed tomography (μCT) on a μCT 40 imaging system (Scanco Medical AG, Bassersdorf, Switzerland) providing an isotropic resolution of 20 μm. A constrained Gaussian filter was used to partly suppress the noise in the volumes. The mineralized bone tissue was differentially segmented from the nonmineralized tissues by using a global thresholding procedure. All samples were binarized using the same parameters for the filter width (1.2), the filter support (1) and the threshold (224; in permille of maximal image gray value).^41,42

Histology and histomorphometry

After μCT imaging, the entire femur was decalcified with 10% ethylene diaminetetraacetic acid (EDTA). The orientation and alignment of the femoral bones were carefully taken into consideration during paraffin embedding to be able to view the defect. Longitudinal serial sections (6-μm thick) were stained with hematoxylin and eosin (H&E) for general histology and histomorphometry.

Using the Image Pro 6 image analysis software (Media Cybernetics, Silver Spring, MD), the area of newly formed bone in the CSD was measured on the H&E-stained sections, and the results were expressed as a proportion (percentage) of the area of the newly formed bone and the area of the original CSD. The results were determined from five H&E-stained sections of the femur of each mouse in the four experimental groups. The mean proportions of each experimental group were compared using an unpaired two-tailed Student's t-test, accepting p<0.05 as significant.

Results

Mechanical testing and analysis

The bending stiffness of intact bone was found experimentally to be \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland,xspace}\usepackage{amsmath,amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6} \begin{document}$$E_1I_1 = 2.275e - 3 [ { \rm Nm}^2 ]$$\end{document} , and the bending stiffness of the fixated bone was found to be \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland,xspace}\usepackage{amsmath,amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6} \begin{document}$$E_2I_2 = 2.125e - 3 [ { \rm Nm}^2 ]$$\end{document} . Where E is the product of the elastic modulus of the beam material and I is the area moment of inertia of the beam cross-section.

The maximal deviation of the bone with the fixator from the intact bone was found to be 1.5 micrometers. Therefore, we concluded that introducing the fixator does not dramatically affect the bending stiffness of the bone.

Postsurgery observations

Postsurgery rehabilitation and complete mobility that included load-bearing functionality were confirmed by analyzing ink-labeled foot-prints of the mice after surgery (Fig. 2). The mice fully recovered from the surgery and displayed a full range of hind leg movements when walking on a treadmill, as shown in a movie taken 10 days postsurgery (Supplementary Movie S1).

FIG. 2.

(A) Ten days postsurgery, showing mobilization and recovery and demonstrating the right hind leg bearing the external fixation device. (B) Footprints recorded with ink staining of the mouse 10 days postsurgery. Color images available online at www.liebertonline.com/tec.

Digital x-ray and μCT

The external fixation frame remained stable, and the edges of the original bone in the CSD were aligned parallel to each other throughout the 8-week study period, data for 0, 4, and 8 weeks are presented. No healing was observed in the empty nongrafted CSD (Fig. 3A–C). Cell-scaffold constructs and scaffold-only x-ray images throughout the experiments are shown in Figure 3D–F and G–I, respectively. Since the scaffold contained mineral (coral/hydroxyapatite), an opaque image was observed in x-ray images from the day of operation and continuously throughout the experiment. This opacity masked the defect site, and the digital x-ray technique was not informative regarding the healing process in these groups. Integration of the graft with the original bone was observed in the CSDs that were filled with the bone allograft (Fig. 3J–L).

FIG. 3.

Digital radiographs of the femur with a nongrafted critical size defect (CSD) of an athymic nude mouse (A) on the day of surgery, (B) 4 weeks after surgery, and (C) 8 weeks after surgery. Digital radiographs of the femur with a CSD that was transplanted with a cell-scaffold construct. (D) on the day of surgery, (E) 4 weeks after surgery, and (F) 8 weeks after surgery. Digital radiographs of the femur with a CSD that was transplanted with a scaffold-only construct. (G) on the day of surgery, (H) 4 weeks after surgery, and (I) 8 weeks after surgery. The ceramic scaffold is opaque and masks the CSD in the digital radiographs (D–G, H, I). Digital radiographs of the femur with a CSD that was transplanted with a bone allograft. (J) on the day of surgery, (K) 4 weeks after surgery, and (L) 8 weeks after surgery. No closure of the defect was observed in the nongrafted CSD (A–C). Integration of the bone allograft with the original bone can be observed in the CSD (J–L).

Examination of the μCT images confirmed no closure of the CSD in the nongrafted CSD (Fig. 4A–C). Examination of the μCT images confirmed integration of the graft in the CSDs that were filled with the bone allograft (Fig. 4D–F); Bone trabeculae were observed between the two edges filling the gap of the original defect.

FIG. 4.

(A) Digital radiographs, (B) microcomputed tomography (μCT) images, and (C) histological micrographs of a nongrafted CSD in the femur, 8 weeks after surgery. (D) Digital radiographs, (E) μCT images, and (F) histological micrographs of femur with a CSD that was transplanted with a bone allograft, 8 weeks after surgery. No closure of the defect is observed in the digital radiographs and the μCT images of the nongrafted CSD, 8 weeks after surgery. No closure and no repair of the CSD are observed in the histological micrograph of the nongrafted CSD. Bone integration is observed in the digital radiographs (D) and μCT (E) images of bone allograft, 8 weeks after surgery. Note smooth integration of the allograft to the host bone (arrow on D, E). An integration of the implant in the original site of the defect is also shown in the histology section of bone allograft, 8 weeks after surgery (F). Note bone trabeculae and continuous cortices on both sides of the bone (arrow on F). The histological sections were stained with hematoxylin and eosin (H&E). Color images available online at www.liebertonline.com/tec.

X-ray images of the cell-scaffold construct and the scaffold only (Fig. 5A, D) revealed the scaffold that contained mineral (coral/hydroxyapatite). The opaque material was also seen in μCT images from both groups (Fig. 5B, E) that were taken at the end of the 8-week study period. To distinguish between the mineral content of the scaffold and the newly formed bone, samples were demineralized and then histologically evaluated.

FIG. 5.

(A) Digital radiographs, (B) μCT images, and (C) histological micrographs of a CSD that was transplanted with a scaffold-only construct and (D) digital radiographs, (E) μCT images, and (F, G) histological micrographs of a CSD that was transplanted with a cell-scaffold construct in the femur of an athymic nude mouse, 8 weeks after surgery. Opaque material is observed in the digital radiographs (A, D) and in the μCT images (B, E). Examination of the histological micrographs revealed no bone formation (except small scattered fractions) in the CSD that was transplanted with a scaffold-only construct (C). Examination of the histological micrograph of the CSD that was transplanted with a cell-scaffold construct in the femur of an athymic nude mouse, 8 weeks after surgery reveal newly formed bone (arrow) (F). Note numerous osteoblasts and osteocytes, shown in a higher magnification of the newly formed bone (G). The histological sections were stained with H&E. Color images available online at www.liebertonline.com/tec.

Histology

Examination of the histology sections of the femurs in which no graft was placed in the CSD confirmed no repair of the CSD with newly formed bone after 8 weeks, the CSD contained connective and adipose tissue (Fig. 4C).

Examination of the histology sections of the femurs in which a bone allograft was placed in the CSD confirmed integration of the graft with the original bone and repair of the CSD with newly formed bone after 8 weeks (Fig. 4F). Examination of the histology sections of the femurs transplanted with cell-scaffold constructs (Fig. 5F) revealed the creation of a bone bridge (in six out of eight animals) in the middle of the bone defect as shown by histology. Higher magnification showed numerous osteoblasts and osteocytes that reside in the newly formed bone (Fig. 5G). In scaffold-only transplants no bone was observed, apart from a few bone fragments (Fig. 5C).

Histomorphometry of the newly formed bone in the CSD area revealed maximal bone formation in the bone allograft (95%), whereas minimal bone formation (10%) was observed in the nongrafted CSD that may be attributed to limited outgrowth from the bone stubs. Histomorphometry of the CSDs that were transplanted with the cell-scaffold construct revealed a substantial amount of newly formed bone (65%) in all CSDs, and a bony bridge was present in six of the eight CSDs, 8 weeks after surgery. Histomorphometry of the CSDs that were transplanted with the noncellular scaffold construct revealed a modest amount of newly formed bone (20%) and no bony bridges in the CSDs (Fig. 6).

FIG. 6.

Quantitative analysis of the area of new bone formation in the CSD of athymic nude mice, 8 weeks after surgery. The data are expressed as the mean±standard deviation of the area of the newly formed bone, in proportion to the total defect area, in the CSD of eight athymic nude mice of each experimental group. *p<0.05, and is the significance of the difference to that of the nongrafted CSD.

Discussion

Various approaches have been used for treating large bone defects²⁸ in the field of tissue engineering. There are several established and acceptable animal models, such as rabbits,^43–46 sheep,^28,47–49 and dogs,^14,50 which have been used for assessing the efficacy of cell therapy based on BMSCs to repair long bone defects. However, these animal models are not available in most research laboratories. In the last few years, CSD models in laboratory rodents became popular for determining the potential of different biomaterials or approaches for bone tissue engineering. The main small animal model in existence, relevant to this type of study, is the immunodeficient rat,^14,27,51 whereas the only available strain is the athymic rat, which is not fully immunocompetent as compared with immunodeficient mice strains available for human cell studies.

Cheung et al. have developed a custom-made external fixation method for repairing femoral fractures in the mouse.⁵² This application was specifically designed for repairing bone fracture and not for creating a segmental bone defect, because the distance between the rods did not allow the creation of a 2–3 mm defect. In addition, its utility was limited because of the need to fabricate a customized bone fixation device.

Several mouse CSD models with internal fixation of the long bone have recently been published.^26,38 In these models, the femur was fixed using a pin that was inserted through the knee, passed through the medulla of the bone, and then fixed by insertion into the supracondylar metaphysic part of the bone.

A defect whose size did not exceed 5 mm was then created in the bone. These models have several limitations, as the insertion of the pin removes a large part of the bone marrow. The surgical procedure involves the fixation of the whole leg and can affect the mobility of the mouse. The method of fixation can affect the weight-bearing aspect of this model Moreover, the inserted pin needs to be completely removed before the mechanical testing, and this removal can cause deformation to the bone that can affect the results of the mechanical tests.

Another CSD model that is sometimes used is the mouse ulnar defect model, where the radius serves as a fixator^53,54 and no external fixation device is used. Despite the simplicity of this model, it has some limitations. First, the model does not truly mimic the human situation of load-bearing. Second, the diameter of the mouse radius is less than 1 mm, and this small size is restrictive, because it does not allow scaffold constructs to be assayed. Third, any repair may be due to a contribution from the adjacent radius.

The external fixation device, which is described in this article, provides a rigid frame for creating a CSD in mice. Our results clearly show that no repair was observed in the untreated femoral CSD, 8 weeks after its creation, and this finding suggests that this CSD is a genuine femoral CSD.^55–57

The CSD model fixated using the external fixation device described in this article mimics the true load-bearing situation needed for clinical bone repair that is crucial for osteogenesis.^19,23 Mechanical testing of the described external fixation device shows that the bending stiffness of the fixated femur is similar to that of an intact femur. Combined with the fact that the overall weight of the device is less than 4% of the mouse body weight, we conclude that this device does not interfere with mobilization after surgery, as seen using the footprints labeling technique and treadmill. Moreover, based on mechanical testing performed on several fixated legs, the model proves to be highly reproducible. It thus provides a reliable load-bearing model in an orthotopic site such as the femur.

It is now more than a decade since the demonstration of ectopic bone formation and subsequent new bone formation using a ceramic scaffold that was loaded with xenogeneic cells in an immunodeficient mouse.^39,58 The new external fixation device, which is described in this article, will allow for the in vivo testing of bone substitute materials impregnated with xenogeneic cells after creating a femoral CSD to be more feasible and accessible to researchers studying bone tissue engineering. The advantages of the model are (1) the fixator is not custom made, and researchers can fabricate and apply it in their laboratories, (2) the volume of the induced femoral CSD is about 15mm³, and this volume should allow investigation of osteoinductivity and osteoconductivity of different BMSC-impregnated scaffolds, and (3) mechanical testing is made feasible, because there is no need to remove the rods that hold the two parts of the femur.

As just described, the femur of nude mice is very small, typically under 15 mm length and 3 mm in diameter. We have, nevertheless, succeeded in demonstrating that it is possible to apply a simple external fixation device to the mouse femur. In the present study, a cell-scaffold construct was used for testing the repair of a femoral CSD in mice. Our results show that 65% of the defect gap volume was filled with newly formed bone in femoral CSD that was transplanted with a cell-scaffold construct. In six out of the eight animals of this experimental group, a bony bridge was observed in the femoral CSD, 8 weeks after surgery, whereas no bony bridge was observed and only 20% of the gap was filled in transplants containing scaffold only. This result indicates the need for osteoconductive biomaterial combined with cells for substantial in vivo bone formation.^59,60 Biocompatible scaffolds are crucial for the in vivo testing of implanted cell-scaffold constructs, because they provide the necessary support for cell proliferation and differentiation.⁸ The results of the present study show that this model can be used for screening of a variety of combinations of cell-based transplant materials, which include various cell types and three-dimensional biomaterials. Such combinations can be screened using this model to optimize protocols for successful in vivo assays. Using this model could be useful for predicting the outcome of testing in larger animals and influence the choice of an experimental protocol in a human clinical study.

In conclusion, the model was shown to be easy and convenient to apply, reproducible, mechanically stable, and light weight. In addition, the model can be used in numerous research settings, such as bone tissue engineering and for studying bone healing in wild-type, immunodeficient, and genetically engineered mice.

Footnotes

Acknowledgments

The authors wish to acknowledge EU grant GENOSTEM (LSH-2003-503161) for supporting this research. The excellent technical assistance of Janette Zavin and Pessia Shenzer is also acknowledged.

Disclosure Statement

No competing financial interests exist.

References

Hutmacher

D.W.

Scaffolds in tissue engineering bone and cartilage. Biomaterials, 21:2529. 2000.

Leong

N.L.

, Jiang

, Lu

H.H.

Polymer-ceramic composite scaffold induces osteogenic differentiation of human mesenchymal stem cells. Conf Proc IEEE Eng Med Biol Soc, 1:2651. 2006.

Livingston

, Ducheyne

, Garino

In vivo evaluation of a bioactive scaffold for bone tissue engineering. J Biomed Mater Res, 62:1. 2002.

Meinel

, Karageorgiou

, Fajardo

, Snyder

, Shinde-Patil

, Zichner

, Kaplan

, Langer

, Vunjak-Novakovic

Bone tissue engineering using human mesenchymal stem cells: effects of scaffold material and medium flow. Ann Biomed Eng, 32:112. 2004.

Meinel

, Karageorgiou

, Hofmann

, Fajardo

, Snyder

, Li

, Zichner

, Langer

, Vunjak-Novakovic

, Kaplan

D.L.

Engineering bone-like tissue in vitro using human bone marrow stem cells and silk scaffolds. J Biomed Mater Res A, 71:25. 2004.

Barrilleaux

, Phinney

D.G.

, Prockop

D.J.

, O'Connor

K.C.

Review: ex vivo engineering of living tissues with adult stem cells. Tissue Eng, 12:3007. 2006.

Dubruel

, Unger

, Vlierberghe

S.V.

, Cnudde

, Jacobs

P.J.

, Schacht

, Kirkpatrick

C.J.

Porous gelatin hydrogels: 2. In vitro cell interaction study. Biomacromolecules, 8:338. 2007.

Srouji

, Kizhner

, Livne

3D scaffolds for bone marrow stem cell support in bone repair. Regen Med, 1:519. 2006.

Srouji

, Maurice

, Livne

Microscopy analysis of bone marrow-derived osteoprogenitor cells cultured on hydrogel 3-D scaffold. Microsc Res Tech, 66:132. 2005.

10.

Ducheyne

, Qiu

Bioactive ceramics: the effect of surface reactivity on bone formation and bone cell function. Biomaterials, 20:2287. 1999.

11.

Ishaug

S.L.

, Crane

G.M.

, Miller

M.J.

, Yasko

A.W.

, Yaszemski

M.J.

, Mikos

A.G.

Bone formation by three-dimensional stromal osteoblast culture in biodegradable polymer scaffolds. J Biomed Mater Res, 36:17. 1997.

12.

Ohgushi

, Caplan

A.I.

Stem cell technology and bioceramics: from cell to gene engineering. J Biomed Mater Res, 48:913. 1999.

13.

Hasegawa

, Neo

, Tamura

, Fujibayashi

, Takemoto

, Shikinami

, Okazaki

, Nakamura

In vivo evaluation of a porous hydroxyapatite/poly-DL-lactide composite for bone tissue engineering. J Biomed Mater Res A, 81:930. 2007.

14.

Bruder

S.P.

, Kurth

A.A.

, Shea

, Hayes

W.C.

, Jaiswal

, Kadiyala

Bone regeneration by implantation of purified, culture-expanded human mesenchymal stem cells. J Orthop Res, 16:155. 1998.

15.

Srouji

, Kizhner

, Suss-Tobi

, Livne

, Zussman

3-D nanofibrous electrospun multilayered construct is an alternative ECM mimicking scaffold. J Mater Sci Mater Med, 19:1249. 2008.

16.

Srouji

, Livne

Bone marrow stem cells and biological scaffold for bone repair in aging and disease. Mech Ageing Dev, 126:281. 2005.

17.

Datta

, Pham

Q.P.

, Sharma

, Sikavitsas

V.I.

, Jansen

J.A.

, Mikos

A.G.

In vitro generated extracellular matrix and fluid shear stress synergistically enhance 3D osteoblastic differentiation. Proc Natl Acad Sci U S A, 103:2488. 2006.

18.

Holtorf

H.L.

, Sheffield

T.L.

, Ambrose

C.G.

, Jansen

J.A.

, Mikos

A.G.

Flow perfusion culture of marrow stromal cells seeded on porous biphasic calcium phosphate ceramics. Ann Biomed Eng, 33:1238. 2005.

19.

M.C.

, Hu

, Zou

S.J.

, Chen

H.Q.

, Zhou

H.X.

, Han

L.C.

Mechanical strain induces osteogenic differentiation: Cbfa1 and Ets-1 expression in stretched rat mesenchymal stem cells. Int J Oral Maxillofac Surg, 37:453. 2008.

20.

Fang

T.D.

, Nacamuli

R.P.

, Song

H.J.

, Fong

K.D.

, Shi

Y.Y.

, Longaker

M.T.

Guided tissue regeneration enhances bone formation in a rat model of failed osteogenesis. Plast Reconstr Surg, 117:1177. 2006.

21.

Loboa

E.G.

, Fang

T.D.

, Parker

D.W.

, Warren

S.M.

, Fong

K.D.

, Longaker

M.T.

, Carter

D.R.

Mechanobiology of mandibular distraction osteogenesis: finite element analyses with a rat model. J Orthop Res, 23:663. 2005.

22.

Loboa

E.G.

, Fang

T.D.

, Warren

S.M.

, Lindsey

D.P.

, Fong

K.D.

, Longaker

M.T.

, Carter

D.R.

Mechanobiology of mandibular distraction osteogenesis: experimental analyses with a rat model. Bone, 34:336. 2004.

23.

Morgan

E.F.

, Longaker

M.T.

, Carter

D.R.

Relationships between tissue dilatation and differentiation in distraction osteogenesis. Matrix Biol, 25:94. 2006.

24.

Fong

K.D.

, Nacamuli

R.P.

, Loboa

E.G.

, Henderson

J.H.

, Fang

T.D.

, Song

H.M.

, Cowan

C.M.

, Warren

S.M.

, Carter

D.R.

, Longaker

M.T.

Equibiaxial tensile strain affects calvarial osteoblast biology. J Craniofac Surg, 14:348. 2003.

25.

Gonzalez

, Fong

K.D.

, Trindade

M.C.

, Warren

S.M.

, Longaker

M.T.

, Smith

R.L.

Fluid shear stress magnitude, duration, and total applied load regulate gene expression and nitric oxide production in primary calvarial osteoblast cultures. Plast Reconstr Surg, 122:419. 2008.

26.

Drosse

, Volkmer

, Seitz

, Penzkofer

, Zahn

, Matis

, Mutschler

, Augat

, Schieker

Validation of a femoral critical size defect model for orthotopic evaluation of bone healing: a biomechanical, veterinary and trauma surgical perspective. Tissue Eng Part C Methods, 14:79. 2008.

27.

Jager

, Sager

, Lensing-Hohn

, Krauspe

The critical size bony defect in a small animal for bone healing studies (II): implant evolution and surgical technique on a rat's femur. Biomed Tech (Berl), 50:137. 2005.

28.

Cancedda

, Mastrogiacomo

, Bianchi

, Derubeis

, Muraglia

, Quarto

Bone marrow stromal cells and their use in regenerating bone. Novartis Found Symp, 249:133; discussion 143, 170, 239,2003.

29.

Kanczler

J.M.

, Ginty

P.J.

, White

, Clarke

N.M.

, Howdle

S.M.

, Shakesheff

K.M.

, Oreffo

R.O.

The effect of the delivery of vascular endothelial growth factor and bone morphogenic protein-2 to osteoprogenitor cell populations on bone formation. Biomaterials, 31:1242. 2010.

30.

Bruder

S.P.

, Jaiswal

, Ricalton

N.S.

, Mosca

J.D.

, Kraus

K.H.

, Kadiyala

Mesenchymal stem cells in osteobiology and applied bone regeneration. Clin Orthop Relat Res, 355,Suppl:S247. 1998.

31.

Chen

, Kidder

L.S.

, Lew

W.D.

Osteogenic protein-1 induced bone formation in an infected segmental defect in the rat femur. J Orthop Res, 20:142. 2002.

32.

Chen

, Schmidt

A.H.

, Tsukayama

D.T.

, Bourgeault

C.A.

, Lew

W.D.

Recombinant human osteogenic protein-1 induces bone formation in a chronically infected, internally stabilized segmental defect in the rat femur. J Bone Joint Surg Am, 88:1510. 2006.

33.

Little

D.G.

, McDonald

, Bransford

, Godfrey

C.B.

, Amanat

Manipulation of the anabolic, catabolic responses with OP-1, zoledronic acid in a rat critical defect model. J Bone Miner Res, 20:2044. 2005.

34.

Schmidhammer

, Zandieh

, Mittermayr

, Pelinka

L.E.

, Leixnering

, Hopf

, Kroepfl

, Redl

Assessment of bone union/nonunion in an experimental model using microcomputed technology. J Trauma, 61:199. 2006.

35.

Shen

H.C.

, Peng

, Usas

, Gearhart

, Fu

F.H.

, Huard

Structural and functional healing of critical-size segmental bone defects by transduced muscle-derived cells expressing BMP4. J Gene Med, 6:984. 2004.

36.

Vogelin

, Brekke

J.H.

, Jones

N.F.

[Heterotopic and orthotopic bone formation with a vascularized periosteal flap, a matrix and rh-BMP-2 (bone morphogenetic protein) in the rat model]

Mund Kiefer Gesichtschir, 4,Suppl 2:S454. 2000.

37.

Vogelin

, Jones

N.F.

, Huang

J.I.

, Brekke

J.H.

, Lieberman

J.R.

Healing of a critical-sized defect in the rat femur with use of a vascularized periosteal flap, a biodegradable matrix, and bone morphogenetic protein. J Bone Joint Surg Am, 87:1323. 2005.

38.

Jacobson

J.A.

, Yanoso-Scholl

, Reynolds

D.G.

, Dadali

, Bradica

, Bukata

, Puzas

E.J.

, Zuscik

M.J.

, Rosier

, O'Keefe

R.J.

, Schwarz

E.M.

, Awad

H.A.

Teriparatide therapy and beta-tricalcium phosphate enhance scaffold reconstruction of mouse femoral defects. Tissue Eng Part A, 17:389. 2010.

39.

Mankani

M.H.

, Kuznetsov

S.A.

, Fowler

, Kingman

, Robey

P.G.

In vivo bone formation by human bone marrow stromal cells: effect of carrier particle size and shape. Biotechnol Bioeng, 72:96. 2001.

40.

Ben-Ari

A.G.V.

, Silbermann

, Sela

, Stein

An external fixation device for immobilization for expeimental fractures in tibiae of rats. J Orthop Surg Tech, 6:47. 1991.

41.

Lutolf

M.P.

, Weber

F.E.

, Schmoekel

H.G.

, Schense

J.C.

, Kohler

, Muller

, Hubbell

J.A.

Repair of bone defects using synthetic mimetics of collagenous extracellular matrices. Nat Biotechnol, 21:513. 2003.

42.

Ruegsegger

, Koller

, Muller

A microtomographic system for the nondestructive evaluation of bone architecture. Calcif Tissue Int, 58:24. 1996.

43.

Fialkov

J.A.

, Holy

C.E.

, Shoichet

M.S.

, Davies

J.E.

In vivo bone engineering in a rabbit femur. J Craniofac Surg, 14:324. 2003.

44.

Stubbs

, Deakin

, Chapman-Sheath

, Bruce

, Debes

, Gillies

R.M.

, Walsh

W.R.

In vivo evaluation of resorbable bone graft substitutes in a rabbit tibial defect model. Biomaterials, 25:5037. 2004.

45.

Tatebe

, Nakamura

, Kagami

, Okada

, Ueda

Differentiation of transplanted mesenchymal stem cells in a large osteochondral defect in rabbit. Cytotherapy, 7:520. 2005.

46.

Saito

, Suzuki

, Kitamura

, Ogata

, Yoshihara

, Masuda

, Ohtsuki

, Tanihara

Repair of 20-mm long rabbit radial bone defects using BMP-derived peptide combined with an alpha-tricalcium phosphate scaffold. J Biomed Mater Res A, 77:700. 2006.

47.

Kon

, Muraglia

, Corsi

, Bianco

, Marcacci

, Martin

, Boyde

, Ruspantini

, Chistolini

, Rocca

, Giardino

, Cancedda

, Quarto

Autologous bone marrow stromal cells loaded onto porous hydroxyapatite ceramic accelerate bone repair in critical-size defects of sheep long bones. J Biomed Mater Res, 49:328. 2000.

48.

Lill

C.A.

, Hesseln

, Schlegel

, Eckhardt

, Goldhahn

, Schneider

Biomechanical evaluation of healing in a non-critical defect in a large animal model of osteoporosis. J Orthop Res, 21:836. 2003.

49.

Marcacci

, Kon

, Zaffagnini

, Giardino

, Rocca

, Corsi

, Benvenuti

, Bianco

, Quarto

, Martin

, Muraglia

, Cancedda

Reconstruction of extensive long-bone defects in sheep using porous hydroxyapatite sponges. Calcif Tissue Int, 64:83. 1999.

50.

Kadiyala

, Young

R.G.

, Thiede

M.A.

, Bruder

S.P.

Culture expanded canine mesenchymal stem cells possess osteochondrogenic potential in vivo and in vitro. Cell Transplant, 6:125. 1997.

51.

Jager

, Sager

, Knipper

, Degistirici

, Fischer

, Kogler

, Wernet

, Krauspe

[In vivo and in vitro bone regeneration from cord blood derived mesenchymal stem cells]

Orthopade, 33:1361. 2004.

52.

Cheung

K.M.

, Kaluarachi

, Andrew

, Lu

, Chan

, Cheah

K.S.

An externally fixed femoral fracture model for mice. J Orthop Res, 21:685. 2003.

53.

Iris

, Zilberman

, Zeira

, Galun

, Honigman

, Turgeman

, Clemens

, Gazit

Molecular imaging of the skeleton: quantitative real-time bioluminescence monitoring gene expression in bone repair and development. J Bone Miner Res, 18:570. 2003.

54.

Moutsatsos

I.K.

, Turgeman

, Zhou

, Kurkalli

B.G.

, Pelled

, Tzur

, Kelley

, Stumm

, Mi

, Muller

, Zilberman

, Gazit

Exogenously regulated stem cell-mediated gene therapy for bone regeneration. Mol Ther, 3:449. 2001.

55.

Hollinger

J.O.

, Kleinschmidt

J.C.

The critical size defect as an experimental model to test bone repair materials. J Craniofac Surg, 1:60. 1990.

56.

Schmitz

J.P.

, Hollinger

J.O.

The critical size defect as an experimental model for craniomandibulofacial nonunions. Clin Orthop Relat Res, 299. 1986.

57.

Schmitz

J.P.

, Schwartz

, Hollinger

J.O.

, Boyan

B.D.

Characterization of rat calvarial nonunion defects. Acta Anat (Basel), 138:185. 1990.

58.

Krebsbach

P.H.

, Kuznetsov

S.A.

, Satomura

, Emmons

R.V.

, Rowe

D.W.

, Robey

P.G.

Bone formation in vivo: comparison of osteogenesis by transplanted mouse and human marrow stromal fibroblasts. Transplantation, 63:1059. 1997.

59.

Bianco

, Robey

P.G.

Stem cells in tissue engineering. Nature, 414:118. 2001.

60.

Robey

P.G.

, Kuznetsov

S.A.

, Riminucci

, Bianco

Skeletal (mesenchymal) stem cells for tissue engineering. Methods Mol Med, 140:83. 2007.

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