Animal protocols were carried out in accordance with Stanford University and VA animal care and use guidelines. Cell sorting, bioconstruct preparation, VML injury, and bioconstruct transplantations were performed previously described,5 briefly summarized below.

Animals

C57BL/6, mice were obtained from Jackson Laboratory. All experimental mice employed were 3–6 months old. Mice were housed and maintained in the Veterinary Medical Unit at the Veterans Affairs Palo Alto Health Care Systems. Animal protocols were approved by the Administrative Panel on Laboratory Animal Care of Stanford University.

Primary cell sorting and bioconstruct preparation

Bioconstructs were prepared by injecting primary cells into scaffolds. We isolated primary cells from skeletal muscles of 3-months-old wild-type male mice (C57Bl/6, Jackson Laboratories) using chemical digestion followed by fluorescence-activated cell sorting. We collected satellite cells, endothelial cells, hematopoietic cells, and fibro-adipogenic progenitors. A mixture was made by combining cells with extracellular matrix proteins to form a hydrogel. Evans blue dye was included in the mixture to enable visualization during injection into the scaffold. Scaffolds were mouse tibialis anterior (TA) muscles that were decellularized with a series of detergents. The mixture was injected into the scaffold using a syringe pump.

VML injury and transplantation surgery

Host mice (NOD/SCID, Taconic) were divided into four treatment groups. The uninjured control group received no-VML injury or treatment. The untreated VML group was injured as previously described.5 Briefly, VML injury was performed by removing rectangular piece of muscle tissue (~15 mg) from the TA. The skin was sutured closed over the wound. For the treatment groups (scaffold only or scaffold plus cells), bioconstructs were generated respectively with scaffold only or with scaffold plus cells as previously described.15 The two treatment groups were injured the same way, and the bioconstructs were sutured into the wound with two proximal sutures and one distal suture into the tendon. The medial and lateral edges of the muscle were sutured closed over the bioconstruct. The skin was sutured closed over the muscle. Surgeries for muscle ablation and bioconstruct implantation were performed blinded by different investigators. The bioconstruct fabrication procedure is standardized by the use of micropumps and micromanipulators.

In vivo force measurement

One month after surgery (age-matched for the uninjured control group), forces in the TA muscles were measured in vivo and ex vivo. For in vivo measurements, mice were anesthetized and the sciatic nerves were placed into bipolar nerve cuffs made with stainless steel wire. To access the sciatic nerve, an incision was made in the skin on the lateral surface of the upper leg. The fascia connecting the hamstrings and vastus lateralis was clipped, and the muscles were retracted to reveal the sciatic nerve. A strip of parafilm was placed under the nerve and stimulator to electrically isolate the surrounding muscles from the stimulator.

The distal tendon of the TA muscle was attached to a force transducer (Aurora Scientific) using suture and a stainless steel wire. The tendon was sutured to one end of the wire and the force transducer was attached to the other end of the wire. To prevent suture slippage during active muscle force, the tendon was first sandwiched between two pieces of wooden toothpick with superglue. The suture was knotted around the tendon proximal to the wooden pieces and then knotted onto a hook at the end of the wire. No suture loops or other significant sources of compliance were present in the system.

We measured viscoelastic force relaxation, passive force, and active twitch force across a range of muscle lengths. A step increase in length of 0.5 mm was applied to the muscle, and the viscoelastic force relaxation trace was recorded for 15 s. We fit a two-phase decay equation to the force relaxation curve and found time constants τ 1 and τ 2 at each length:

$$P = P_0 + P_1{\mathrm{e}}^ \wedge ( - \tau ^ \wedge - \tau _1) + P_2{\mathrm{e}}^ \wedge ( - \tau ^ \wedge - \tau _2).$$

The measurement was repeated at each muscle length. The mean value of τ 1 and τ 2 across trials at different muscle lengths is reported for each TA.

After 1 min of relaxation, bipolar twitch stimuli were applied via the nerve cuff (6 twitches, 1 Hz, 1 ms stimulus duration). The baseline force before each twitch was subtracted from the maximum force during the twitch to calculate active twitch force. The three highest twitch forces were averaged at each length. The baseline force before the final twitch was recorded as the passive force at that length. Thus we measured active and passive forces across a range of lengths in 0.5 mm increments. The three maximal twitches at the optimal length were further analyzed for time from stimulus to peak force and time to relax from peak force to half of the peak force. The force-length curve was normalized to maximum twitch force and offset to length at maximum twitch force.

After completing the length-tension curve with twitches (2 increments of 0.5 mm beyond the optimal), the muscle was returned to optimal length and stimulated at increasing frequencies (60, 80, 100, 120, and 140 Hz) to determine the maximum tetanic force. Maximum twitch force was plotted against muscle mass as an indirect measurement of active specific tension. Length at optimal force was measured with digital calipers as the distance from the fibular head to the muscle-tendon junction.

Both TAs were tested. After the first TA was tested, the lower leg was wrapped in parafilm with PBS while the contralateral TA was tested. Limb order was randomized. Preliminary testing in C57Bl6 mice showed no difference between TAs tested first or second. An Aurora Scientific 1300-A Whole Mouse Test System was used to gather force production data. Animals were killed by cervical dislocation under anesthesia.

Ex vivo force measurement

To measure the force independent of innervation, we isolated the TA in a bath of oxygenated Ringer’s solution and stimulated it with plate electrodes. Immediately after euthanasia, the distal tendon of the TA, the TA, and the knee (proximal tibia, distal femur, patella, and associated soft tissues) were dissected out and placed in Ringer’s solution (Sigma) maintained at 25 °C with bubbling oxygen with 5% carbon dioxide. The proximal tibia was sutured to a rigid wire attached to the force transducer and the distal tendon was sutured to a rigid fixture. No suture loops or slack was present in the system. The contralateral limb was immediately dissected and kept under low passive tension in oxygenated Ringer’s solution bath until measurement. Supramaximal stimulation voltage was found and the active force-length curve was measured in a manner similar to the in vivo condition. After measurement, the muscle was dissected free and the mass measured. An Aurora Scientific 1300-A Whole Mouse Test System was used to gather force production data.

Image analysis

Image J was used to calculate the percentage of area composed of Collagen by using the color threshold plugin to create a mask of only the area not occupied by transplanted scaffold and positive for Collagen. That area was then divided over the total area of the sample which was found using the free draw tool.

Statistical analysis

A second-order regression curve was fit to the in vivo and ex vivo length-tension curves of each group and the second-order term compared with an f-test. The passive force-length relationship was compared with linear regression between the lengths of L o −0.5 to L o +0.5. The active and passive curves of the VML group treated with scaffold plus cells were pairwise compared to those from each of the other groups. Differences among the means of optimal length, time to peak twitch force, time from twitch peak to half relaxation, and viscoelastic time constants were compared with one-way ANOVA with post hoc Dunnett pairwise comparison between the VML group treated with scaffold plus cells and each of the other groups. To evaluate potential differences in the active stress generation among the groups, we plotted maximum ex vivo tetanic force vs mass and used linear regression to test the null hypothesis that one line describes all the groups. To analyze collagen area quantification data, two-sided test was used.

Immunostaining

A 1-h blocking step with 20% donkey serum/0.3% Triton in PBS was used to prevent unwanted primary antibody binding for all samples. Primary antibodies were applied and allowed to incubate over night at 4 °C in 20% donkey serum/0.3% Triton in PBS. After 4 washes with 0.3% PBST, fluorescently conjugated secondary antibodies were added and incubated at room temperature for 1 h in 0.3% PBST. After three additional rinses each slide was mounted using Fluoview mounting media.

Imaging

Samples were imaged using standard fluorescent microscopy (Zeiss Observer Microscope Inverted Motorized Fluorescence Phase Contrast) and either a ×10 or ×20 air objective. Volocity imaging software was used to adjust excitation and emission filters and came with pre-programmed AlexaFluor filter settings which were used whenever possible. All exposure times were optimized during the first round of imaging and then kept constant through all subsequent imaging.

Antibodies

The following antibodies were used in this study. The source of each antibody is indicated: Collagen I (Cedarlane Labs, #CL50151AP, 1:200); Laminin (Millipore, #MAB1903, 1:750).

General methods

Unless stated otherwise, sample size (n-values) are reported as biological replicates of mice and/or SC isolations from separate mice performed on different days. In most cases, the data presented were compiled over the course of 3 years, as mice with the appropriate genotype became available. Therefore, the magnitude of the effect and variability in the measurements were primary factors in determining sample size and replication of data. Although samples were not explicitly randomized or blinded, mouse identification numbers were used as sample identifiers and thus the genotypes and experimental conditions of each mouse/sample were not readily known or available to the experimenters during sample processing and data collection. The only criteria used to exclude samples involved the health of the animals, such as visible wounds from fighting. In these cases, the animals were handled in accordance with approved IACUC guidelines.

The research was conducted in accordance to all relevant guidelines and procedures