An Inexpensive 3D Printed Mouse Model of Successful, Complication-free Long Bone Distraction Osteogenesis

Abstract

Distraction osteogenesis (DO) is used for skeletal defects; however, up to 50% of cases exhibit complications. Previous mouse models of long bone DO have been anecdotally hampered by postoperative complications, expense, and availability. To improve clinical techniques, cost-effective, reliable animal models are needed. Our focus was to develop a new mouse tibial distractor, hypothesized to result in successful, complication-free DO.

Methods: A lightweight tibial distractor was developed using CAD and 3D printing. The device was fixed to the tibia of C57Bl/6J mice prior to osteotomy. Postoperatively, mice underwent 5 days latency, 10 days distraction (0.15 mm every 12 hours), and 28 days consolidation. Bone regeneration was examined on postoperative day 43 using micro-computed tomography (μCT) and Movat's modified pentachrome staining on histology (mineralized volume fraction and pixels, respectively). Costs were recorded. We compared cohorts of 11 mice undergoing sham, DO, or acute lengthening (distractor acutely lengthened 3.0 mm).

Results: The histological bone regenerate was significantly increased in DO (1,879,257 ± 155,415 pixels) compared to acute lengthening (32847 ± 1589 pixels) (P < 0.0001). The mineralized volume fraction (bone/total tissue volume) of the regenerate was significantly increased in DO (0.9 ± 0.1) compared to acute lengthening (0.7 ± 0.1) (P < 0.001). There was no significant difference in bone regenerate between DO and sham. The distractor was relatively low cost ($11), with no complications.

Conclusions: Histology and µCT analysis confirmed that the proposed tibial DO model resulted in successful bone formation. Our model is cost-effective and reproducible, enabling implementation in genetically dissectible transgenic mice.

Fig. 1.

Development of a lightweight distractor using computer-aided design and 3D printing. Oblique view of tibial distractor plates with length (x), width (y), and height (z) of distractor plate computer-aided design measured in inches.

Fig. 2.

Tibial distractor placement—surgical technique. A, Preparation of surgical site (shaving and aseptic preparation) followed by lower limb external rotation with incision made from knee to ankle. B, Dissection of tibialis anterior muscle. C, Drill hole at tibial crest shown with white arrowhead, and drill hole at the mid-shaft shown with black arrowhead. D, Left to right: Distractor, distraction screw, and tibial fixation screws. E, Proximal distractor plate shown in situ. F, Distal distractor plate shown in situ. G, Transverse tibial osteotomy creation (broken black line). H, Dental drill equipment: (left to right) 0.6-mm drill bit, Medium MiniFlex Double-Sided Diamond Disc, 3.175-mm chuck (CHK-3.175 91593, Nakanishi Inc., Tokyo, Japan), and 2.35-mm chuck (H203A180A, Brasseler Dental Instrumentation, Ga.). I, Distractor screw in situ with fracture reduction, adequate alignment, and subsequent skin closure. Scale bar on all panels represents 3.5 mm.

Fig. 3.

Tibial distractor—osteogenic validation. A, Mouse tibial distractor schematic. B, Schematic describing protocol timeline for (top to bottom) gradual distraction, acute lengthening, and sham control. The final time point was 43 days postoperatively for all cohorts. C, Left to right: Representative μCT and pentachrome stained section of the sham tibia at POD 43. On pentachrome staining, bone appears yellow, cartilage appears blue-green, muscles appear bright red, and stroma appears brown. Scale bar for all histology images represents 100 µm. D, Left to right: Representative μCT and pentachrome stained section of the acute lengthened tibia at POD 43. E, Left to right: Representative μCT and pentachrome stained section of the gradually distracted tibia at POD 43. F, Graph demonstrating the pixel density of bone following histomorphometric analysis of micrographs of sections obtained from (left to right) sham, acute lengthening, and distraction, which were stained using Movat’s pentachrome. G, Graph demonstrating the percentage of bone volume/tissue volume for (left to right) sham, acute lengthening, and distraction, which was analyzed using μCT.