Minced muscle autografting improves bone healing but not muscle function in a porcine composite injury model

Composite tissue injuries (CTIs) in extremities include segmental bone defects (SBDs) and volumetric muscle loss. The objective of this study was to determine if skeletal muscle autografting with minced muscle grafts (MMGs) could improve healing in an SBD and improve muscle function in a porcine CTI model that includes an SBD and adjacent volumetric muscle loss injury. Adult Yucatan Minipigs were stratified into three groups including specimens with an isolated SBD, an SBD with volumetric muscle loss (CTI), and an SBD with volumetric muscle loss treated with MMG (CTI + MMG). Bone healing was quantified with serial x‐rays and postmortem computed tomography scanning. Muscle function was quantified with a custom in vivo force transducer. Muscle tissue content was determined by biochemical analyses and histology. Anterior cortex‐modified Radiographic Union Score for Tibia fractures (mRUSTs) decreased from 2.7 to 1.9 (p = 0.003) in CTI versus SBD animals. MMG improved anterior mRUST scores to 2.5 in CTI + MMG specimens (p = 0.030 compared to CTI specimens) and overall mRUST scores increased from 9.4 in CTI specimens to 11.1 in CTI + MMG specimens (p = 0.049). Residual strength deficits at euthanasia were 42% in SBD (p < 0.001), 44% in CTI (p < 0.001), and 48% in CTI + MMG (p < 0.001) compared to preoperative values. There were no differences in strength deficits between the three groups. Biochemical and histologic analyses demonstrated scattered differences between the three groups compared to contralateral muscle. MMG improved bone healing. However, the primary cause of muscle dysfunction and biochemical changes was the presence of an SBD. Clinical significance: Early mitigation of SBDs may be necessary to prevent muscle damage and weakness in patients sustaining composite extremity trauma.


| INTRODUCTION
High-energy extremity trauma frequently results in segmental bone defects (SBDs), with severe adjacent skeletal muscle damage, up to and including volumetric muscle loss (VML). SBDs can lead to severe functional limitations and amputation. [1][2][3] VML results in permanent weakness and pain. 4 Bone healing within these composite tissue injuries (CTIs) depends on a competent adjacent soft-tissue envelope. Animal models have consistently demonstrated that overlying compromised skeletal muscle impairs bone healing. [5][6][7][8][9] Similarly, clinical studies confirm that skeletal muscle-based defect coverage restores underlying bone healing. 10 As such, clinical treatment algorithms for CTIs involve initial wound debridement, skeletal stabilization, soft-tissue stabilization, and wound coverage. VML defects are typically treated by rotational or remote tissue transfers to cover SBDs, restoring vascularity, and ushering in immune-based cells that promote bone healing and prevent infection. However, rotational and remote muscle tissue transfers do not restore function to the affected musculature and thus the weakness associated with VML remains unaddressed.
Accordingly, researchers are working toward developing interventions for VML that provide wound coverage and restore muscle function.
Two examples of treatments for VML defects include the implantation of biologic scaffolds 11 and minced skeletal muscle autograft (MMG). 12 Pollot et al. 11 showed that implantation of a biologic scaffold into a VML defect neither improved muscle function nor improved fracture healing in a rat CTI model. MMG implantation, however, enhanced fracture healing and improved neuromuscular function in rats with tibial osteotomies and adjacent VML. 12 Furthermore, MMG dampened the heightened infiltration of immune cells into the SBD, characteristic of extremities with VML, that impairs fracture healing in rodents. 6 All of these reports are based on observations in rodents and need to be confirmed in a higher order species. To that end, we have shown that MMG has improved histological outcomes with concomitant improvements in neuromuscular function in a porcine-isolated VML model. [13][14][15] In other work, we have developed an isolated porcine SBD model, which results in slow but consistent healing of a 25.0 mm defect in the mid-diaphysis of the tibia. 16,17 The purpose of this study was to combine our VML and SBD injury models into a porcine CTI model in Yucatan Minipigs (YMPs).
The experiment was designed to (1) determine how VML affects bone healing in an SBD and (2) determine if MMG improves bone healing and/or muscle function in an SBD with adjacent VML. We hypothesized that VML would impair the healing of an SBD and that treatment of the VML injury with MMG would rescue bone healing and improve the neuromuscular function of porcine limbs with a CTI.

| Experimental design
Three groups of YMPs (n = 7/group) were studied, including: (1) pigs with an isolated 25.0 mm SBD to the mid-diaphysis of the tibia (SBD), (2) pigs with the SBD and an adjacent untreated VML injury (CTI), and (3) pigs with the CTI and the VML defect treated with MMG (CTI + MMG). Specimens were euthanized 3 months after injury.
Bone healing was quantified with serial x-rays at monthly intervals and confirmed with computed tomography (CT) scanning postmortem. In vivo functional muscle testing of both legs was performed just before surgery and at monthly intervals until euthanasia. Postmortem muscle samples were collected for biochemical and histologic analyses.

| Animals and surgical treatment
Adult castrated male YMPs 16-20 months old (Sinclair BioResources) were acclimated in individual cages with ad lib access to water and feeding. Animals were induced with an intramuscular injection of telazol (500 mg), xylazine (250 mg), and ketamine (250 mg). After endotracheal intubation, isoflurane was titrated (0.25%-5% mixed with 100% oxygen) throughout the operation based on vital signs, jaw tone, and withdrawal reflexes. Hydromorphone (0.05 mg/kg) was delivered intravenously before incision.
Central vascular access was established in the external jugular vein.
YMPs were positioned ventral side up and the right lower extremity was prepared with sterile methods. The mid-diaphysis of the tibia was exposed through a 12.0-cm curvilinear anterior incision beginning 3.0 cm below the knee joint and 5.0 mm lateral to the tibial tubercle. 16,17 The incision coursed distal and curved medially approximately 1.0 cm lateral to the subcutaneous tibial border. The anterior compartment musculature was dissected away from the tibia exposing the mid-diaphysis. A 25.0-mm SBD was mapped out at the beginning 5.0 mm inferior to the distal end of the tibial tubercle. Subsequently, a 7-hole 3.5-mm dynamic compression plate (Stryker Orthopaedics) was contoured to fit the lateral surface of the tibia and secured above and below the SBD site with two 3.5 mm screws. Next, a 7-hole 1/3rd tubular plate was secured on the medial surface spanning the planned resection site with two 3.5 mm screws. The medial plate was removed, and the lateral plate loosened allowing access to the tibial osteotomy sites. Two parallel transverse cuts were made with a reciprocating saw and the bone was resected. The lateral plate was retightened, and the medial plate was reapplied. Three additional screws were inserted in the lateral plate (for a total of three above the SBD and two below the SBD) and two additional screws were inserted into the medial plate (for a total of two screws above and two below the SBD).
These methods maintained the dimensions of the SBD and anatomic alignment of the tibia ( Figure 1A). 16,17 Subsequently, in the CTI and CTI + MMG groups, 7.0 g of muscle, adjacent to the SBD, was sharply resected from a 3.0 cm x 3.0 cm footprint centered in the peroneus tertius (PT) muscle ( Figure 1B).
The muscle was incrementally resected from the entire footprint until 7.0 g was removed. The VML defect was approximately 10.0 mm deep and purposefully was never full-thickness with approximately 10 mm of muscle remaining between the SBD and the base of the VML defect.
In CTI + MMG animals, 5.25 g of the resected muscle was manually minced in a sterile test tube with surgical scissors into small (<1.0 mm 3 ) pieces and placed back into the VML defect ( Figure 1C). The fascia overlying the filled defect was closed with

| X-ray bone healing
Bone healing was quantified using fluoroscopic radiographs made at monthly intervals until euthanasia. Radiographs were evaluated by three of the investigators who were blinded to the groups. The modified Radiographic Union Score for Tibia Fractures (mRUSTs), which assigns an integer score from 1 to 4 to each of the four cortices (anterior, posterior, medial, and lateral), was used. 18,19 The integer scores are as follows: 1 = no callus; 2 = callus present but not bridging F I G U R E 1 (A-C) Schematic depiction of the three experimental groups. Isolated segmental bone defect (SBD) animals (A; left) had a 25.0 segment of mid-diaphyseal bone cut from the tibia and the defect was stabilized with two plates. The adjacent anterior compartment musculature was not injured. In composite tissue injury (CTI) animals (B; middle), the same SBD was made and stabilized with dual plating and a 7.0-g volumetric muscle loss defect was created in the anterior compartment adjacent to the SBD. Note the volumetric muscle loss (VML) was not full thickness and the underlying SBD and lateral plate were covered with intact muscle. The footprint of the defect was 3.0 cm × 3.0 cm. In the CTI + MMG animals (C; right), the same SBD and VML defects were created and the VML defect was filled with 5.3 g of minced muscle autograft (MMG).

| CT-based bone healing
After euthanasia, tibias were dissected and hardware removed for CT evaluation using a high-resolution CT scanner (XtremeCT II; Scanco Medical). The scanner operated at 68 kVp and 1.47 mA with a 60-ms integration time to acquire a stack of 816 tomographic slices with a voxel size of 50 µm. The slice stack was centered in the middle of the SBD. The reconstructed tomographic images were imported into OsiriX MD 11.0.3 (Pixmeo SARL) to create sagittal and coronal plane reconstructions spanning the defect site. Two-dimensional sagittal and coronal plane images were used to determine the presence or absence of bone bridging involving the medial, lateral, anterior, and posterior cortices.

| In vivo muscle strength testing
In vivo muscle functional assessments were performed under anesthesia to assess the strength of the dorsiflexor muscles using a custom-fabricated large animal force transducer (Model 892A; Aurora Scientific) as previously described. 13 Testing was repeated at a range of ankle positions from 0°to 40°p lantarflexion, in 10°increments, with 2 min rest between tests. We recorded the maximum torque value detected at any one of the ankle positions for each specimen. Furthermore, protein lysates were analyzed for hydroxyproline content to quantify collagen. 22 The assay was performed by adding 12 N HCl to soluble peptides and proteins in each standard and tissue sample to hydrolyze to individual amino acids by incubating at 100°C for 3 h, followed by 18 h incubation at 95°C. Samples were allowed to cool to room temperature and then suspended in deionized water.

| Muscle biochemical analyses
Hydroxyproline amino acids were converted to pyrolle-2-carboxylate by oxidation via the addition of 50 µl chloramine-T solution, followed by incubation at room temperature for 20 min. Finally, 50 µl p-dimethylaminobenzaldehyde in 2-propanol plus perchloric acid was added and vortexed immediately to facilitate mixing. Samples were incubated at 60°C for 30 min and rapidly cooled by immersion in room temperature water to stop chromophore development. The plate was read in a spectrophotometer at an absorbance wavelength between 550 and 565 nm.

| Muscle histologic analyses
PT muscles were fixed in 10% neutral-buffered formalin and processed for paraffin embedding. Cross-sections (6 μm) were prepared and stained using standard protocols for picrosirius red (Abcam) and for trichrome images. Brightfield images were acquired with a Zeiss Axio Scan.Z1 and qualitative assessments of morphology and composition were made by observing 4-6 muscles per group.
Quantitative analyses were performed on the indexed image values after global thresholding and segmentation in MATLAB (Mathworks). Analyses were performed using random intercepts for observers.

| Statistical analyses
Significance was determined by a p ≤ 0.05. In addition, χ 2 analyses were performed between the three groups specifically comparing pigs with healing trajectories ( CTI + MMG) demonstrate successful healing in four SBD animals, two CTI animals, and five CTI + MMG animals. There are distinct trajectory differences between the two CTI animals that healed compared to the five CTI animals that did not heal (B, dashed oval). Overall modified Radiographic Union Score for Tibia fracture (mRUST) scores demonstrate that minced muscle grafting improved overall bone healing (D) at 3 months after injury in CTI + MMG pigs (mRUST = 11.1 ± 2.8) compared to CTI pigs (mRUST = 9.4 ± 2.6) (*denotes a significant increase in CTI + MMG pigs compared to CTI pigs, p = 0.049). Specimens with successful healing trajectories (overall mRUST ≥ 10 at 3 months, mRUST increase ≥ 1 in the 2-to 3-month interval) are denoted in dashed rectangles in (A-C) compared to specimens with failed healing trajectories (mRUST < 10) in dashed triangles. χ 2 analyses demonstrated that CTI + MMG pigs had significant improvements in overall healing trajectories compared to CTI pigs (**between B and C denotes improved healing trajectories in CTI + MMG specimens compared to CTI specimens with p = 0.048). MMG, minced muscle graft. 0.88. For the entire duration, our interrater coefficient was 0.86, demonstrating excellent concordance between the raters.

| VML impaired fracture healing in the anterior cortex of the SBD
Overall mRUST scores were consistently lower in CTI specimens relative to SBD specimens, but the differences were not statistically significant (p = 0.18) ( Figure 2D). Individual healing trajectories demonstrated that four of five SBD animals had a progression of bone healing in the 3rd month (Figure 2A). In contrast, five of seven CTI animals had mRUST ≤ 10 in the 3rd month ( Figure 2B) (p = 0.08).
Impaired bone healing in the CTI animals was largely in the anterior cortex adjacent to the VML ( Figure 3A). At 3 months, VML reduced radiographic bone healing in the anterior cortex from an mRUST of 2.7 in SBD specimens to 1.9 (p = 0.003) in CTI specimens. This healing difference accounted for the flattened overall mRUST healing trajectories ( Figure 2D) in the CTI animals in the 3rd month.

| MMG improves bone healing in an SBD
At 3 months after injury, overall mRUST scores were 9.4 in CTI specimens and 11.1 in CTI + MMG specimens (p = 0.049) ( Figure 2D).
Five of six CTI + MMG specimens were on a positive healing trajectory in month 3 ( Figure 2C) in contrast to CTI specimens that had failed healing trajectories in five of seven specimens (p = 0.048) ( Figure 2B). At 3 months postinjury, MMG resulted in an increase in healing in the adjacent anterior cortex, which increased from 1.9 in CTI specimens to 2.5 in CTI + MMG specimens (p = 0.030; Figure 3A).

| Radiographic bone healing results were confirmed on CT scanning
In the CTI specimens, two of seven specimens had complete cortical bridging and five of seven were unhealed with no bridging cortical bone (Supporting Information: Figure 1). Four of six of the CTI + MMG specimens exhibited complete cortical bridging, and a fifth specimen was nearly bridged with <1 mm gap (Supporting Information: Figure 2). (p < 0.001) ( Figure 4B)

| Muscle biochemistry
There were no differences in any biochemical markers ( Figure 5A-I) between the three experimental groups and total protein ( Figure 5J) was similar between all three groups and contralateral samples. The three markers of myogenesis MYH1, MYH2, and MYH3 ( Figure 5D-F) trended lower in all groups and were significantly decreased in SBD animals compared to contralateral limbs. The fibrosis marker CTGF was increased in CTI animals ( Figure 5H). Interestingly, the neuroinnervation markers CHRNA and TUBB3 were increased in the CTI + MMG group (Figures 5A,B) compared to contralateral samples.

| Muscle histology
Muscle samples from the CTI group exhibited heightened collagen matrix deposition relative to the uninvolved contralateral limbs (p = 0.01) ( Figure 6). Fibrosis is depicted in both picrosirius ( Figure 6A) and trichrome ( Figure 6B) sections. No differences in collagen fraction were observed between SBD, CTI, and CTI + MMG samples.

| DISCUSSION
The principal findings of this study are twofold: (1)  | 1897 bone healing, 9,24 and surgical amelioration of the VML defect with MMG restored bone healing.
The second principal finding of this study was that the presence of an SBD negatively impacts the adjacent skeletal muscle regardless of the injury status of that muscle. This finding was unanticipated and incongruent with findings from analogous injury models in rodents. 6,9,11,12,25 In a rat CTI study by Willett et al., 5 muscle strength was significantly decreased in animals with isolated bone injuries (analogous to our SBD group). However, strength deficits in this model were twice as severe in rats with combined bone and muscle injury (analogous to our CTI group) versus an isolated bone injury.
These data parallel the findings of Hurtgen et al. 12 that demonstrated strength deficits were doubled in rats with combined bone and muscle injury compared to isolated bone injury. We anticipated that treatment with MMG would partially restore muscle architecture and improve function in CTI injuries, as has been previously shown in an isolated (i.e., no bone injury) porcine VML model. 13

| Limitations
There are several experimental discrepancies that could have affected bone healing. Two SBD and one CTI + MMG specimens had catastrophic hardware failure within 2 weeks of surgery. None of the specimens could protect their weight bearing after surgery and it is much more likely that the early failures were due to an excessive torsional load during standing as opposed to early impaired bone healing. 16,17 One pig in both the CTI group and CTI + MMG group developed a postoperative wound infection. Both wounds resolved with local debridement and both pigs healed their fracture, so it is unlikely that the infections affected differences in bone healing observed between the groups. We used the mRUST method to quantify healing. Multiple studies have shown that this method is subjective with notable inter-and intrarater variability. 18,19 However, our interrater variability in this study, and in our previous work, 16,17 were consistent with the previously published ranges. Finally, mRUST data were confirmed with CT scanning.
Changes in muscle torque were likely affected by discrepancies in electrode placement to stimulate the common peroneal nerve.
Depolarization was reproducible in intact legs without injury.
However, in postinjury testing, optimizing electrode placement was often difficult to obtain maximum muscle torque. Frequently, we had to parametrically adjust electrode placement until we observed a reliable waveform that depicted successful stimulation. Judgment was made by inspecting the upslope (typically ≤2-3 ms) and waveform during the 800 ms maximal stimulation pulse (≤2%-3% attenuation of magnitude during the pulse) of the muscle contraction (Supporting Information: Figure 4). Additionally, disuse resulting from injury invariably contributed to muscle weakness and we did not formally quantify postinjury activity. However, animals that healed F I G U R E 5 (A-J) Violin plots of biochemical markers harvested from segmental bone defect (SBD) (n = 4), composite tissue injury (CTI) (n = 6), and CTI + MMG (n = 6) specimens depict the expression of various protein markers associated with myogenic, fibrogenic, and neurogenic processes measured in samples taken from the belly of the peroneus tertius muscles adjacent to the SBD. Samples from uninjured contralateral muscle (n = 4 each from SBD, CTI, and CTI + MMG for a total of 12 samples) were obtained as control muscle. Note that total protein content (J) was similar between experimental groups and contralateral samples. Myogenic proteins in all three groups (D-F) trended lower than the contralateral samples with significant reductions in SBD animals in all three myogenic components and a reduction in MYH3 in CTI + MMG animals. Two markers of neuroinnervation, nicotinic acetylcholine receptor (CHRNA) and tubulin β3 chain (TUBB3), were increased in CTI + MMG animals compared to contralateral specimens. However, there were no differences in the myogenic proteins between the three groups (*indicates a difference in the mean of the annotated group relative to samples from the uninvolved contralateral limbs, p < 0.05). MMG, minced muscle graft. Trichrome staining (B) further illustrates extensive fibrosis in all three groups compared to contralateral samples. Fibrotic tissue, quantified and presented as the percentage of the total cross-sectional area of the tissue samples, was found to be increased and myogenic tissue percentage was decreased in samples from the composite tissue injury (CTI) group relative to the samples from the uninvolved contralateral limbs (C). No difference in fibrotic tissue or myogenic tissue composition of samples taken from the involved limbs was observed between experimental groups (*indicates a difference in the mean of the annotated group relative to samples from the uninvolved contralateral limbs, p < 0.01). MMG, minced muscle graft; SBD, segmental bone defect.
their SBDs were typically weight-bearing without any noticeable limp by the 3rd month. Accordingly, regional effects of bone injury on muscle function and composition were likely mediated by factors beyond disuse.

| CONCLUSIONS
In summary, MMG improved bone healing in an SBD with adjacent VML. SBDs caused fibrosis and weakness in the adjacent injured and uninjured muscles. MMG did not restore muscle function in a VML defect.