Rabbit Model of Physeal Injury for the Evaluation of Regenerative Medicine Approaches

Abstract

Physeal injuries can lead to bony repair tissue formation, known as a bony bar. This can result in growth arrest or angular deformity, which is devastating for children who have not yet reached their full height. Current clinical treatment involves resecting the bony bar and replacing it with a fat graft to prevent further bone formation and growth disturbance, but these treatments frequently fail to do so and require additional interventions. Novel treatments that could prevent bone formation but also regenerate the injured physeal cartilage and restore normal bone elongation are warranted. To test the efficacy of these treatments, animal models that emulate human physeal injury are necessary. The rabbit model of physeal injury quickly establishes a bony bar, which can then be resected to test new treatments. Although numerous rabbit models have been reported, they vary in terms of size and location of the injury, tools used to create the injury, and methods to assess the repair tissue, making comparisons between studies difficult. The study presented here provides a detailed method to create a rabbit model of proximal tibia physeal injury using a two-stage procedure. The first procedure involves unilateral removal of 25% of the physis in a 6-week-old New Zealand white rabbit. This consistently leads to a bony bar, significant limb length discrepancy, and angular deformity within 3 weeks. The second surgical procedure involves bony bar resection and treatment. In this study, we tested the implantation of a fat graft and a photopolymerizable hydrogel as a proof of concept that injectable materials could be delivered into this type of injury. At 8 weeks post-treatment, we measured limb length, tibial angle, and performed imaging and histology of the repair tissue. By providing a detailed, easy to reproduce methodology to perform the physeal injury and test novel treatments after bony bar resection, comparisons between studies can be made and facilitate translation of promising therapies toward clinical use.

Impact Statement

This study provides details to create a rabbit model of physeal injury that can facilitate comparisons between studies and test novel regenerative medicine approaches. Furthermore, this model mimics the human, clinical situation that requires a bony bar resection followed by treatment. In addition, identification of a suitable treatment can be seen in the correction of the growth deformity, allowing this model to facilitate the development of novel physeal cartilage regenerative medicine approaches.

Introduction

The physis, or growth plate, is a cartilage structure located at the end of all long bones in children, and functions as the primary site of bone elongation. It is also a weaker area of the developing skeleton, given its juxtaposition between stiff bones. Approximately 18–30% of pediatric fractures involve the physis, and although many heal without negative sequalae, an estimated 10–30% of these physeal fractures will develop bony repair tissue at the injury site, forming what is known as a bony bar.^1–3 Complications resulting from bony bars include angular deformities or complete growth arrest. Such outcomes are devastating for young children who have not yet reached their full height, and can in turn lead to an abnormal gait, low back pain, and early-onset osteoarthritis.

Current clinical treatments focus on prevention methods and are unable to regenerate the injured physis. Once the bony bar forms, it is commonly resected and replaced with an interpostional material such as fat or cement.^4–6 The material serves as a placeholder while the surrounding healthy physeal cartilage maintains bone elongation. Unfortunately, this approach is not always successful, and the bony bar often reforms and negatively affects growth.⁷ There is a critical need to develop effective treatments for physeal injuries that can prevent bone formation after bony bar resection and also lead to physeal cartilage regeneration. For this reason, various regenerative medicine approaches for the treatment of pediatric physeal injuries are being developed, and include cell-based therapies, growth factors, and biomaterials.⁸ To test the efficacy of these potential treatments, animal models that emulate human physeal injury are necessary.

Mouse and rat physeal injury models, where a drill-hole defect is created in the femoral or tibial physis, have provided insight into the mechanisms of bony bar formation and have allowed various biological treatments to be tested for its prevention.^9–11 The limitation of these small animal models is that the bony bar that forms cannot easily be resected and replaced with an interpositional material as is performed clinically. To do so, larger animals such as the rabbit, miniature pig, and sheep have been used.^12–14 In these models, physeal injury leads to a bony bar that can be resected, followed by application of a potential treatment. Outcomes such as the effect of the therapy on bone lengthening and angular deformity can be tracked, as well as bony repair tissue formation. The rabbit has been commonly used for this purpose: partial removal of the physis leads to bony repair tissue formation within 3 weeks, followed by resection of the bony bar, and application of an interpositional material.

Various methods are currently used to induce physeal injury in the rabbit (Table 1). The distal femur and proximal tibia are the most common locations for physeal injury, although the distal ulna has also been reported. Although some studies report removing 10% of the physis, others remove >50%. The tools used to make the injury also vary and include a scalpel, drill bit, bur, or curette. Another major difference to be noted is that although most rabbit studies of physeal injury applied a treatment after bony bar resection, in some instances treatments were applied immediately after injury. Outcomes such as bone lengthening, changes in angular deformity, and histological analysis were not reported in all studies, although they provide the most complete set of information on the effect of the treatment applied. Overall, there is a need to establish a reproducible animal model of physeal injury to facilitate comparisons between studies, for testing novel regenerative medicine approaches, and to identify therapeutics that warrant further investigation for the clinical treatment of pediatric physeal injuries.

Table 1.

Summary of Experimental Studies Using a Rabbit Model of Physeal Injury to Test Regenerative Medicine Therapies

Reference No.	Location of physeal injury	Area of physis removed	Tools used to create physeal injury	Time treatment was delivered	Control groupa(C)	Outcomes
						Limb Length	Angular deformity	CT/MRI	Histology
13	Distal femur	67%	Unknown	Immediately after injury	Blood clot implant (C)	√			√
15	Distal femur	>9%	3.5 mm drill bit	3 weeks postinjury	Untreated (C)	√	√	√	√
16	Distal femur	50%	Cylindrical trocar	Immediately after injury	Untreated (C)		√		√
17	Proximal tibia	33%	1.0 mm burr	3 weeks postinjury	Intact (C)		√	√	√
18	Proximal tibia	3.0 or 1.2 mm diameter, 5.0 mm depth	3.0 and 1.2 mm drill tool	(No treatment) observation	Intact (C)	√		√	√
19	Distal femur	35%	4.5 mm drill tool	Immediately after injury	Intact (C)	√			√
20	Distal ulna	Unknown	2.0 mm drilling tool and chisel	Immediately after injury	Intact (C)				√
21	Proximal tibia	67%	No. 15 scalpel blade, curette	Immediately after injury	Untreated (C)	√	√		√
22	Proximal tibia	50%	5.0 mm burr, microcurette	Immediately after injury	Untreated (C)	√	√	√	√
23	Proximal tibia	50%	21G needles, curettes	Immediately after injury	Untreated	√	√		√
14	Proximal tibia	25%	Trephine needle	3 weeks postinjury	Intact (C)				√
24	Proximal tibia	4.0 mm diameter, 3.0 mm depth	COR system	Immediately after injury	Untreated (C)		√		√
25	Proximal tibia	50%	2.0 mm dental burr	Immediately after injury	Untreated		√		√
26	Proximal tibia	40%	A specially designed box osteotome	Immediately after injury	Untreated (C)			√	√
27	Distal femur	50%	Sagittal saw	Immediately after injury	Intact (C)	√			√
28	Proximal tibia	50%	No. 21G needles and curettes	Immediately after injury	Sham surgery (C)	√	√		√
29	Proximal tibia	50%	Scalpel	Immediately after injury	Untreated	√	√		√
30	Proximal tibia	>50%	Unknown	3 weeks postinjury	Untreated	√	√		√
31	Distal femur	33%	Oscillating saw	Give indomethacin immediately before injury	Saline injection	√	√		√
32	Proximal tibia	50%	1.0 mm burr	3 weeks postinjury	Untreated (C)				√
33	Proximal tibia	50%	High-speed steel burr	Immediately after injury	Untreated		√	√	√
34	Distal femur	5.0 mm diameter centrally	5.0 mm drill bit	Immediately after injury	Untreated (C)			√	√

^aUntreated: physeal injury is left empty; intact: no procedure was performed; sham surgery: incision was made and then closed without physeal injury.

C, contralateral; CT, computed tomography; MRI, magnetic resonance imaging; COR, consistent osteochondral repair.

The aim of this study was to present methods for a rabbit model of proximal tibial physeal injury, using a staged procedure. The first step involves a physeal injury and the second step involves bony bar resection and treatment. In this study, we tested the implantation of a fat graft that is used clinically after bony bar resection and a photopolymerizable hydrogel as a proof of concept that injectable materials could be delivered into this type of injury. These two treatments were compared with animals whose resection site was left untreated. The different outcomes used to evaluate the repair tissue and effect of treatment on bone elongation and angular deformity are discussed.

Materials and Methods

Figure 1 provides an overview of the different surgical procedures that are performed to create a physeal injury and test a therapy, as well as the outcomes used to evaluate the repair tissue.

FIG. 1.

Flow chart of the study design with the two surgical procedures and outcomes used to evaluate repair.

Animal preparation

All procedures were in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the University of Colorado Denver. Thirty-two 6-week-old New Zealand white rabbits (21 males and 11 females; Charles River Laboratories, Inc., Wilmington, MA) were purchased and allowed a 1-week acclimatization period before planned procedures. They were given food and water ad libitum. On the day of surgical procedures, rabbits were anesthetized with intravenous ketamine (25 mg/kg) and xylazine (2.5 mg/kg) and maintained with 1.5–3.0% inhaled isoflurane through a nose cone. Rabbits were laid supine on a heating pad, both eyes were lubricated (Optixcare Eye Lube; Adventix, Seattle, WA) and vital signs were monitored continuously during anesthesia. Surgical areas were shaved and scrubbed with iodine, 70% ethanol, and then sterilely draped. The ipsilateral foot was wrapped in a sterile surgical glove.

Proximal tibia physeal injury

An initial pilot study with 3 rabbits and a second study with 29 rabbits were performed. Rabbits were subjected to an induced physeal injury in their right tibia. A 3 cm anterior-medial longitudinal incision, slightly medial to the tibial tubercle was performed. Soft tissue was dissected and the medial collateral ligament (MCL) distal insertion was identified and served as a landmark. The physis was easily distinguished from the surrounding bone as a white line of tissue between the tibial tubercle and the MCL distal insertion (Fig. 2A, arrow). Its location was further confirmed by inserting 23G needles into the physis and performing intraoperative radiographs (AJEX Meditech Ltd; J type stand unit) (Fig. 2B). Once location and orientation were confirmed, a 1 mm wide burr (Dental Burs USA) attached to a rotary tool (Dremel^®; drill speed 15,000 rpm) was directed perpendicular to the tibial shaft and parallel to the joint line. The depth of the injury was controlled and limited to 5 mm by mounting a plastic stopper to the burr (Fig. 2C). A 5 × 5 × 1 mm defect, ∼25% of the physis, was made (Fig. 2D). A nonabsorbable 5-0 nylon suture knot was placed on the lateral edge of the injury and served as a marker for the second procedure (Fig. 2E, arrow). After saline irrigation of the defect, the fascia and skin were closed using 3-0 Vicryl and 4-0 polydioxanone (PDS) sutures, respectively. The contralateral limb was left uninjured throughout the study. The animals were allowed normal cage activity and exercise. In an initial study, three rabbits were euthanized after 3 weeks to image and measure the bony bar that formed to better plan the surgical resection of the bony bar.

FIG. 2.

Creation of the proximal tibia physeal injury. (A) Identification of the physis (white tissue shown by yellow arrow); (B) 23G needles inserted into the physis to facilitate its location during radiography; (C) plastic stopper placed 5 mm from the end of the burr to control the depth of the injury; (D) a 5 × 5 × 1 mm injury to the physis; (E) nonabsorbable suture (yellow arrow) placed on the lateral edge of the injury served as a landmark for future procedures.

Surgical resection of bony bar

Three weeks after the initial physeal injury, rabbits were anesthetized as described for the first surgery, and a radiograph was taken to confirm formation of a bony bar at the injury site. A 4 cm anterior-medial longitudinal incision was made over the previous incision. The injured area was exposed and the nonabsorbable suture placed during the first surgery was located (Fig. 3A, arrow). The bony bar was localized with a three-way landmark reference: (1) the suture knot; (2) 23G guide needles placed in the knee joint, and in the bony bar; and (3) radiography (Fig. 3B). Two guide needles were inserted into the bony bar: the first was ∼1–2 mm proximal to the suture knot, and the other was 1 mm proximal and 6 mm medial to the suture knot. The guide needle in the knee joint was inserted parallel to the medial tibial plateau. This needle was used as a reference for the orientation of the bony bar.

FIG. 3.

Bony bar resection and treatment procedure. (A) Exposure of injured physis and bony tissue, with arrow pointing to the reference suture knot; (B) location of the bony bar by intraoperative radiography, asterisk shows bony bar; (C, D) bony bar resection using a metal guide to create a resection site of similar shape and size; (E) fat graft placement after bony bar resection; (F) hydrogel injected and polymerized into resection site, asterisk shows the hydrogel in the defect.

Any callus tissue that formed adjacent to the physis and bony bar was removed with a scalpel to expose the bony bar within the physeal area. A rotary tool (Dremel, drill speed 15,000 rpm) with a 1 mm burr was used to resect the bony bar, by removing an area measuring 6 × 6 × 2 mm. To create a consistent resection, a plastic stopper was placed on the burr at a height of 6 mm. A custom-made metal guide measuring 6 × 6 × 2 mm was periodically inserted into the defect to ensure that all resections were the same shape and size (Fig. 3C, D). The area was irrigated with saline, and a gelatin sponge (Jorgensen Laboratories, Inc.) was used to control bleeding. The injured area was dried with gauze and filled according to the following treatment groups: (1) untreated, (2) fat graft (Fig. 3E), or (3) a hydrogel material (Fig. 3F). There were 9–10 rabbits per group. The wound was then closed as previously described.

Fat graft preparation

Animals that were assigned to the fat graft treatment had a fat graft removed from their inguinal area during the surgical resection of the bony bar. A 1 cm oblique incision was made 2 cm cranial and 0.5 cm lateral of the scrotum in male rabbits, and in a correlating location in female rabbits. A 1 cm diameter section of fat tissue was excised under the subcutaneous layer and a saline soaked gauze was applied to cover the incision temporarily. The fat graft was immediately inserted into the bony bar resection site. Once the excised fat tissue sufficiently filled the defect, the skin incision made to harvest the fat graft was closed with 4-0 PDS sutures. Given that the fat retrieved is superficial, there is minimal risk to underlying tissues. However, care should always be taken.

Hydrogel preparation

A previously developed hydrogel for cartilage tissue engineering was used as a proof of concept that injectable materials could be placed into the resection site and remain in place.¹⁵ Eight-arm poly (ethylene glycol) (10 kDa) endcapped with norbornene (PEGNB) was synthesized from eight-arm PEG Amine (JenKem) that was reacted overnight, at room temperature under inert atmosphere with 5-norbornene-2-carboxylic acid with 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate methanaminium and N,N-diisopropylethylamine in dimethylformaide/dichloromethane to give an amido linkage between the PEG Amine and norbornene. The PEGNB product was precipitated in diethylether and then filtered, dialyzed, and lyophilized. The hydrogel was prepared from a solution of PEGNB (9 wt%) and with 50 nM tethered transforming growth factor-β3 (Peprotech, Inc.) and 35 mM matrix metalloproteinase-sensitive peptide crosslinker (GCVPLS-LYSGCG; GenScript) in phosphate-buffered saline.¹⁶ The 0.1 mM cell adhesion peptide GCRGDS (GenScript), 1% (g/g) thiolated chondroitin sulfate (15% thiolated, molecular weight: ∼47kDa), and 0.05% (g/g) lithium phenyl-2,4,6-trimethylbenzoylphosphinate as the photoinitiator were also added to the precursor solution and the solution was sterile filtered. All chemicals were obtained from Sigma-Aldrich unless otherwise noted. Bleeding was controlled in the defect and then the defect was dried before injecting the hydrogel. The white lights in the operating room were turned off and a red lamp was used for hydrogel injection and polymerization to prevent any unintended polymerization. Approximately 70–75 μL of the prepolymer solution was injected into the defect and photopolymerized using a 395–480 nm light source (VALO^® LED Curing Light) for 1–2 min.

Measurement of tibial length and tibial angle

Radiographs of the lower limbs were obtained before the initial physeal injury, at 3 weeks postinjury (at time of surgical resection of the bony bar), and at 8 weeks post-treatment. They were used to measure tibial length and tibial angle. The rabbits were placed supine, and held in place by immobilizing the upper body. The pelvis was positioned within the X-ray field and the two rear paws were pulled to straighten the lower limbs and ensure that the patella was positioned forward in all animals (Fig. 4A). The images were then used to measure tibial length using two methods. Lines were drawn along the tibial plateau (across the very lateral and medial points of the proximal tibia; Fig. 4B, purple line) and along the tibiotalar joint line (Fig. 4B, blue line). A mechanical axis line was drawn along the long axes of the tibia and femur (Fig. 4B, C, red line). First method, length “a,” measures the distance from the tibial plateau line to the tibiotalar joint line (Fig. 4B, green line), whereas the second method, length “b,” measures the distance from the lateral end of the tibial plateau line to the lateral end of the tibiotalar joint line (Fig. 4B, yellow line). The tibial angle was also measured with two different methods: the medial proximal tibial angle (MPTA, α) and the proximal tibial medial plateau angle (PTMPA, β). Lines were drawn along the tibial plateau (Fig. 4C, purple line), and along the medial tibial plateau (across the very top point of tibial eminence and the very medial point of proximal tibia; Fig. 4C, orange line). A mechanical axis line was drawn along the axes of the tibia and femur, and α and β were calculated to be the angles between the mechanical axis line and the tibial plateau line, and medial tibial plateau line, respectively (Fig. 4C).

FIG. 4.

Rabbit positioning for radiographs and methods used to measure tibia length and angular deformity. (A) Positioning of rabbit for radiograph; (B) schematic for tibia length measurement using method “a” and method “b”; (C) schematic for tibial angle measurement using method α and β.

X-ray microscopy

Rabbits were euthanized at 3 weeks after initial physeal injury or at 8 weeks after bony bar resection and treatment. The tibia were harvested, trimmed, and placed in 10% neutral-buffered formalin before imaging. To assess bony bar formation, the samples were imaged with an X-ray microscope (XRM) (Xradia Versa XRM-520; Zeiss, Dublin, CA) using the following parameters: energy of 7 W at 80 kV, voxel size of 24 ± 3 μm, 0.4 × objective, 801 projections using the LE3 filter, exposure of 0.6–0.7 s, and source and detector distances of 40 and 75 mm, respectively. After the scans, 2D images were captured by slicing the 3D volume on the frontal plane to visualize the injury location and repair tissue.

Histology

Tibias were fixed in 10% neutral-buffered formalin under cyclic agitation for 7 days before being processed. Bones were trimmed to remove the tubercle and posterior condyles, rinsed with deionized water, and decalcified in 14% ethylenediaminetetraacetic acid for 6–8 weeks. Samples that were not fully decalcified were transferred to Immunocal (Fisher Scientific) for 1 week. Once decalcified, the tissues were embedded in paraffin, sectioned to 5 μm in the coronal plane, and mounted on positively charged slides. Deparaffinized sections were stained with Alcian Blue Hematoxylin with Orange G/Eosin counterstain (ABH stain), which stains cartilage tissue blue, bone tissue bright orange, fibrous tissue pink, and hematopoietic/marrow tissue dark purple.

Statistical analysis

Data are reported as mean ± standard deviation. Comparison between groups was performed using two-way ANOVA (analysis of variance) with Bonferroni's multiple comparisons test. Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software). A value of p ≤ 0.05 was considered significant.

Results

Proximal tibia physeal injury leads to bony bar formation

In this study, all rabbits that received an injury to the proximal tibial physis developed a bony bar that was visible by radiograph at 3 weeks postinjury (Fig. 3B). Once the skin was opened, the nonabsorbable suture that was placed during the first surgery to serve as a landmark was easily found and facilitated the location of the injured physis (Fig. 3A). Animals euthanized at 3 weeks postinjury had their tibia scanned by XRM, and showed evidence of bony repair tissue within the injured physis, spanning the epiphysis and metaphysis (Fig. 5A, yellow box). The imaging also confirmed that there was a callus formation adjacent to the injured physis in all animals (Fig. 5A, area outlined in orange). The images were used to measure the size of the bony bar that formed, and it was determined that resecting an area measuring 6 × 6 × 2 mm would ensure complete removal of the bony repair tissue (Fig. 5B, yellow box).

FIG. 5.

XRM image of proximal tibia 3 weeks after physeal injury. Bony bar is outlined by the yellow box. Repair callus tissue that formed adjacent to the injury site is outlined in orange. XRM, X-ray microscope.

Proximal tibia physeal injury leads to reduced limb lengthening and angular deformity

Radiographs taken 3 weeks postinjury demonstrated that the injured right tibias were shorter than the contralateral uninjured left tibias and also displayed an angular deformity (Fig. 6A). Tibial length measurements indicated that although both the injured right tibia and uninjured left tibia underwent limb lengthening during the 3 weeks after the injury (Fig. 6B, C, **p < 0.0001), the right tibia was significantly shorter than the uninjured left tibia at 3 weeks postinjury, regardless of whether the measurement was made using method “a” (Fig. 6B, *p < 0.05) or method “b” (Fig. 6C, *p < 0.05). The injured tibia also had a significant angular deformity compared with the uninjured contralateral side at 3 weeks postinjury, and this was evident with both methods used to measure angular deformity (Fig. 6D, E, **p < 0.0001).

FIG. 6.

Effect of physeal injury on limb lengthening and angular deformity. (A) Radiograph of rabbit lower limbs 3 weeks after physeal injury to the proximal tibia showing a shorter tibia and pronounced angular deformity on the injured right side (R) compared with the uninjured left side (L). (B) Tibial length before physeal injury and 3 weeks postinjury using method “a” and (C) using method “b.” (D) Tibial angle before physeal injury and 3 weeks postinjury using method “α” and (E) method “β.” *p < 0.05, **p < 0.0001.

Evaluation of tibial length, tibial angle, and repair tissue at 8 weeks post-treatment

Tibial lengths measured using method “a” at 8 weeks post-treatment were similar in all animals, regardless of whether they received a fat graft (103.7 ± 5.1 mm), hydrogel (101.8 ± 6.3 mm), or were left untreated (102.4 ± 4.4 mm). Measurements using method “b” were also similar between groups (106.4 ± 4.8 mm for untreated, 108.5 ± 4.6 mm for fat graft, and 105.9 ± 5.8 mm for hydrogel). Treatment of the resection site with fat graft or hydrogel did not improve the tibial angle compared with the untreated group (method α: 64.1°± 10 for fat graft and 58.0°± 7.4 for hydrogel compared with 58.4°± 7.1 for untreated; method β: 59.3 ± 8.2 for fat graft and 53.5°± 5.8 for hydrogel compared with 54.3°± 5.5 for untreated). XRM at 8 weeks post-treatment showed an intact physis in the uninjured tibias (Fig. 7A), whereas rabbits that were left untreated after bony bar resection had evidence of recurring bony repair tissue formation (Fig. 7B). Bone tissue was also present in the fat graft group (Fig. 7C) and hydrogel group (Fig. 7D), although it appeared to be less than the untreated group. Histological analysis of the repair tissue showed that the uninjured physis had continuous physeal cartilage (Fig. 7E, I), whereas the untreated group contained a continuous and dense bony bar at the injury site (Fig. 7F, J). The rabbits that received a fat graft showed evidence of bone and a large area of fibrous tissue at the injury site (Fig. 7G, K). Although the animals that received the hydrogel also showed bone formation, there were small areas of cartilage-like tissue surrounding the area where the original injury was (Fig. 7H, white box). Closer magnification of the area shows a mixture of fibrous tissue and hypertrophic chondrocytes (Fig. 7L).

FIG. 7.

Characterization of repair tissue in the proximal tibia 8 weeks post-treatment. (A–D) XRM images (scale bar: 5 mm), (E–H) ABH stain (scale bar: 1000 μm) of the repair tissue and insets are show in (I–L) at higher magnification (scale bar: 100 μm). (A, E, I) Normal (uninjured); (B, F, J) untreated; (C, G, K) fat graft; (D, H, L) hydrogel.

Discussion

This study describes the methodology to create a rabbit model of proximal tibia physeal injury that consistently leads to a bony bar, reduced limb lengthening, and pronounced angular deformity. We also detail the methods to resect the bony bar in a well-controlled manner to effectively evaluate novel regenerative medicine approaches.

Numerous rabbit models of physeal injury have been reported yet vary in terms of size and location of injury and methods to assess repair tissue after treatment (Table 1). Although both the tibia and femur have been used to create physeal injuries that lead to bony bar formation, our group chose to focus on the proximal tibial physis because of its morphology. The distal femoral physis has pronounced peaks and valleys, which can make it challenging to resect the physeal tissue without damaging the surrounding bone from the epiphysis or the metaphysis. The physis in the proximal tibia has a flatter anatomy, which facilitates accurate drilling of physeal tissue.

The size of the initial physeal injury is another important aspect that must be taken into consideration when choosing a model. A rabbit study by Janarv et al. concluded that growth disturbances occurred when the physeal drill injury destroyed at least 7–9% of the distal femoral physis.¹⁷ Cao et al. also compared different sizes of physeal tissue damage, such as 4%, 6%, and 8% of the physeal area, and after 2 weeks they did not observe any abnormal growth, suggesting that injuries affecting <8% of the physis area can still maintain normal bone elongation.¹⁸ Most reported studies using the rabbit model of physeal injury remove 50% of the physeal cartilage (Table 1). Clinically, if the bony bar that forms after physeal injury is <50% of the physis, resection and graft interposition is attempted to prevent bony fusion and diminish growth abnormalities.¹⁹ To lead to bony bar formation and to be clinically relevant, it is recommended that physeal injuries span an area ranging from 8% to 50% of the entire physis. For this reason, we chose to use a 5 × 5 × 1 mm physeal injury, which represents ∼25% of the proximal tibial physis in a 6-week-old rabbit. This led to a consistent bony bar, a reduced limb lengthening, and evident angular deformity within 3 weeks after injury, representing the main outcomes seen in children affected by bony bars.

During all surgical procedures, knowledge of the exact orientation of the physis and location of the bony bar was critical to achieve accurate drilling. The use of intraoperative radiographs and needles to guide location is highly recommended. Higher resolution imaging such as micro computed tomography and XRM at 3 weeks postinjury was helpful in initial studies to better understand the size and location of the bony bar that formed and how to better plan the surgical approach. The use of a metal guide during the resection surgery permitted the surgeon to create a resection site that was of the same size and shape in each animal. The use of a 1 mm dental burr for drilling was found to provide a greater degree of accuracy than a larger burr as it prevented excessive tissue damage. Overall, imaging modalities and use of standardized tools facilitated the surgeries and provided a higher level of consistency between animals, and are highly recommended to perform the procedures associated with this animal model.

Two methods were presented in this study to measure tibial length. Method “a” is a commonly used method to measure tibial length, whereas method “b” was chosen to capture the highest aspect of the tibia despite the angular deformity that occurs after physeal injury. Because both methods indicated a difference between the uninjured and injured limb, both are considered appropriate to measure limb length in this animal model. Two methods were also used to measure tibial angle and capture the extent of angular deformity. The MPTA (α) is the angle between the long axis of the tibia and tibial plateau that reflects the entire plateau deformity.²⁰ In the clinical forum, the MPTA is the standardized radiographic measurement that is used to measure tibial angular deformity. The PTMPA (β) was also investigated as it can provide additional information on how the medial tibial plateau changed after physeal injury. Although it has not been used in the clinical population, the PTMPA did show differences in this animal study and is recommended as a potential measure of angular deformity.

The animal model presented in this study, if left untreated after bony bar resection, will lead to bony bar reformation. This provides an opportunity to test novel regenerative medicine approaches after resection. These potential treatments can be evaluated for their capacity to prevent recurrence of bony bar formation after resection, restore normal bone elongation, improve the angular deformity that resulted from the initial injury, and ultimately to regenerate the physeal cartilage tissue. Implantation of an autologous fat graft, as commonly performed clinically, did not prevent bone formation or promote cartilage regeneration, although more fibrous tissue formed compared with the untreated group. The hydrogel that was used as a proof of concept as an injectable material demonstrated that it could be delivered into the resection site and cured in situ. It also led to reduced bone formation and some evidence of cartilage-like tissue formation, suggesting that its optimization for physeal cartilage regeneration is warranted. Although the fat graft and hydrogel-treated rabbits did not show significant limb lengthening or angular deformity correction at 8 weeks post-treatment, this may be a result of the study endpoint set at 8 weeks, whereas other studies have evaluated outcomes at time points of 16 weeks and up to 52 weeks post-treatment.^21–29 Long-term studies will continue to investigate the hydrogel as a potential regenerative medicine approach for physeal injuries.

Hydrogels are widely studied for the regeneration of articular cartilage in animal models of cartilage injury, with many having shown promising results.^30–34 They are of particular interest in tissue engineering because their physical and chemical properties can be modified to direct stem cell fate and promote the desired tissue.^35–37 The rabbit model of physeal injury described in this study provides an additional model to test the cartilage regeneration potential of various hydrogels currently being used for articular cartilage.

In conclusion, we have provided methodology to induce a physeal injury in the proximal tibia of a 6-week-old New Zealand white rabbit that spans ∼25% of the physis area. This injury led to bony bar formation, limb length discrepancy, and angular deformity in all animals within 3 weeks. We outlined methods to accurately resect the bony bar with the use of imaging modalities and surgical templates. The described animal model of physeal injury provides an opportunity to test novel regenerative medicine treatments, such as hydrogels, that could correct growth deformities. By providing a detailed approach to induce physeal injury and evaluate potential therapies, a comparison between studies can be achieved and can facilitate translation of promising therapies toward clinical use.

Disclosure Statement

No competing financial interests exist.

Funding Information

Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health & Human Development of the National Institutes of Health (NIH) under award number R21HD090696 and by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the NIH under award number 1R01AR069060. Research was also supported by the Gates Grubstake Fund. A Major Research Instrumentation Award from the National Science Foundation (NSF#1726864) supported the purchase of the XRM system.