Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2022 Dec 1.
Published in final edited form as: J Tissue Eng Regen Med. 2021 Sep 27;15(12):1131–1143. doi: 10.1002/term.3243

Co-delivery of fibrin-laminin hydrogel with mesenchymal stem cell spheroids supports skeletal muscle regeneration following trauma

Peter Genovese 1, Anjali Patel 1, Natalia Ziemkiewicz 1, Allison Paoli 1, Joseph Bruns 1, Natasha Case 1, Silviya P Zustiak 1, Koyal Garg 1,*
PMCID: PMC8648985  NIHMSID: NIHMS1742871  PMID: 34551191

Abstract

Volumetric muscle loss (VML) is traumatic or surgical loss of skeletal muscle with resultant functional impairment. Skeletal muscle’s innate capacity for regeneration is lost with VML due to a critical loss of stem cells, extracellular matrix, and neuromuscular junctions. Consequences of VML include permanent disability or delayed amputations of the affected limb. Currently, a successful clinical therapy has not been identified. Mesenchymal stem cells (MSCs) possess regenerative and immunomodulatory properties and their three-dimensional aggregation can further enhance therapeutic efficacy. In this study, MSC aggregation into spheroids was optimized in vitro based on cellular viability, spheroid size, and trophic factor secretion. The regenerative potential of the optimized MSC spheroid therapy was then investigated in a murine model of VML injury. Experimental groups included an untreated VML injury control, intramuscular injection of MSC spheroids, and MSC spheroids encapsulated in a fibrin-laminin hydrogel. Compared to the untreated VML group, the spheroid encapsulating hydrogel group enhanced myogenic marker (i.e., MyoD and myogenin) protein expression, improved muscle mass, increased presence of centrally nucleated myofibers as well as small fibers (<500 μm2), modulated pro- and anti-inflammatory macrophage marker expression (i.e., iNOS and Arginase), and increased the presence of CD146+ pericytes and CD31+ endothelial cells in the VML injured muscles. Future studies will evaluate the extent of functional recovery with the spheroid encapsulating hydrogel therapy.

Keywords: Volumetric muscle loss, extracellular matrix, hydrogels

1. Introduction

Skeletal muscle has a remarkable regenerative capacity following mild or moderate injuries. In severe muscle traumas such as a volumetric muscle loss (VML), the critical-sized defect exceeds the body’s regenerative capacity resulting in chronic loss of function and permanent disability [1]. Initially, the loss of vital regenerative elements such as satellite cells and the basal lamina limit muscle fiber regeneration. Subsequently, a prolonged pro-inflammatory immune response inhibits myogenic pathways while promoting fibrotic tissue deposition [2]. Currently, there are no successful clinical therapies for the treatment of VML. This presents a significant opportunity to develop stem cell or biomaterial-based tissue engineering strategies for VML repair.

Mesenchymal stem cells (MSCs) exhibit attractive features for tissue engineering including ease of isolation, ex vivo expansion capacity, self-renewal, multipotency, immunomodulatory properties, and the secretion of growth factors and cytokines [3]. Over 1,000 completed or ongoing clinical trials utilizing MSCs for a wide range of applications have been investigated [4]. However, the beneficial effects of MSC based therapies in initial small-scale clinical studies are often not corroborated in large clinical trials [5, 6]. Several limitations associated with cell-based therapies have yet to be overcome. The primary obstacles of MSC-based therapies include poor survival due to apoptosis, necrosis, or anoikis, and the inability to maintain self-renewal [7]. Due to the stress of transferring MSCs out of standard culture conditions, damage during the injection process, or the harsh microenvironment of the transplanted site, fewer than ~5% of transplanted MSCs are reported to survive at the site of injection days after transplantation [8, 9]. MSCs transplanted at sites of injury encounter a disrupted extracellular matrix (ECM) as well as a hypoxic and inflamed microenvironment, which may not be conducive for cell survival or renewal [8].

Three-dimensional (3D) aggregate or ‘spheroid’ formation is a promising approach to improve the efficacy of stem cell therapies and achieve tissue-like cell density (107–109 cells/cm3) [10] for transplantation. MSC culture in spheroid form has been shown to augment their stemness, post-transplant survival, anti-inflammatory effects, angiogenic response, and differentiation potential [11]. MSC aggregation is thought to be controlled through intrinsic cell-cell contacts and cell-matrix interactions. Aggregation also enhances secretion of trophic factors and ECM proteins [12], increases cell adhesion/retention, and offers protection from the cytotoxic microenvironment at the injury site [13, 14]. Several preclinical studies have demonstrated the benefits of transplanting MSCs as high-density 3D aggregates. For instance, in hindlimb models of ischemia [15, 16], intramuscular injection of MSC spheroids increased angiogenesis, attenuated necrosis, and showed better proliferation than MSC suspension in the ischemic region. In mouse models of dystrophic and cardiotoxin injured skeletal muscle [17], intramuscular injection of MSC spheroids enhanced muscle function. However, the efficacy of MSC spheroids has never been tested in a VML model.

It has been repeatedly shown that MSCs injected with a biomaterial carrier material – typically a viscous protective ECM solution exhibit better viability and retention in the transplanted tissue [18-20]. Several studies have used biomaterial-based strategies to provide adhesion sites and biomechanical support to transplanted MSCs [8, 9]. These biomaterials are also expected to shield the MSCs from inflammatory cells and cytokines while simultaneously permitting the diffusion of oxygen, nutrients, and waste materials [21]. MSC spheroids have been encapsulated in collagen [22], fibrin, [23] and alginate[24] hydrogels for various tissue engineering applications.

In this study, we chose a well-characterized laminin (LM)-111 enriched fibrin hydrogel for encapsulation of MSC spheroids [25, 26]. LM-111 is a potent activator of satellite cells and can influence their adhesion, migration, proliferation, and differentiation [27]. Besides satellite cells, LM-111 can also modulate MSC activity. LM promotes MSC paracrine activity [28], maintains stemness [29], and suppresses pro-inflammatory function [27].

In our previous studies, LM-111 enriched FBN hydrogels were tested in vitro and implanted in vivo in a murine model of VML in the gastrocnemius-soleus complex [25, 26]. The in vitro study concluded that the corporation of LM-111 (450 μg/ml) in fibrin hydrogels resulted in a biomaterial that matched the mechanical characteristics of muscle tissue and augmented the expression of MyoD and desmin in C2C12 myoblasts [26]. When implanted in a murine model, LM-111 (450 μg/mL) enriched fibrin hydrogels supported cellular infiltration, myogenic protein expression, but improvements in muscle function were not observed [25].

In this work, we develop and optimize an MSC spheroid therapy for skeletal muscle regeneration following VML. We hypothesize that the delivery of MSC spheroids using fibrin enriched LM-111 hydrogels will reconstitute myogenic building blocks (i.e., stem cells and ECM) to enhance skeletal muscle regeneration following VML.

2. Materials and Methods

2.1. Cell Culture and Aggregate Formation

Bone marrow-derived murine MSCs (ATCC, CRL-3220) were cultured in minimum essential medium eagle (α-MEM; Corning) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) for all experiments. MSCs were sorted as CD140b+CD73+CD34−CD45− and used from passage 2-5 for all experiments [30]. Additionally, we have observed that these MSCs also express CD146 and SCA-1 in culture (Supplementary Figure 1A). In vitro differentiation of MSCs into adipocytes, chondrocytes, and osteocytes was confirmed with a stem cell differentiation kit (R&D Systems) [30].

MSCs were cultured in AggreWell™ 400 24-well plates (STEMCELL Technologies) containing an array of 400×400 μm-sized microwells. AggreWell plates were used to ensure consistency in spheroid size. In addition, spheroids can be removed relatively easily from the AggreWell plates without significant damage to the spheroidal structure compared to other methods [31]. Before seeding, the Aggrewell™ plates were treated with 500 μL of anti-adherence rinsing solution (STEMCELL Technologies) and centrifuged at 1300*g for 5 min. The plates were then washed twice with 1 mL of media. MSC monolayers were dissociated with 0.25% trypsin with EDTA (Corning), counted, and then added to the Aggrewell™ plate. MSCs were seeded at a density of 1.8, 3.6 and 7.2 x 105 cells/well to obtain 150, 300, and 600 cells/microwell. The plates were centrifuged at 100 g for 3 min to force cell aggregation into the wells and incubated at 37°C under 5% CO2 for 24 hrs. Spheroids were collected from the plate through pipetting. For the single-cell control, MSCs were seeded at a density of 3.6 x 105 cells/well in a 24 well-plate. Cells were cultured in α-MEM supplemented with 10% FBS and 1% P/S. For in vitro analysis, single cells and spheroids were cultured in media for up to 7 days. To maintain aggregation, Aggrewell™ plates were placed on a rotary shaker at 50 rpm.

2.2. Spheroid Size and Cellular Viability

Brightfield images of the Aggrewell™ plates were taken of the spheroids at days 1, 4, and 7 to assess their size and morphology. Briefly, Images were taken using a Zeiss Axiocam microscope at 10× magnification. Images were taken of 3 different wells (n=21-47 spheroids/group/timepoint). These images were analyzed in Fiji (distribution of the open-source software ImageJ), where the spheroids were manually outlined, and the average of the major and minor axes of the fitted ellipse was taken to be the spheroid diameter.

Live/Dead staining of spheroids harvested from the Aggrewell™ plates was done per manufacturer’s (Invitrogen) instructions at days 1, 4, and 7 of culture. Cell proliferation of MSC spheroids was also tested on days 1, 4, and 7 using the PicoGreen assay (Invitrogen). Aggregates were released by pipetting with media, and then added to a centrifuge tube and centrifuged at 1500 rpm for 5 minutes. The excess media was removed, and the cell pellets were frozen at −20°C. To prepare the cell lysate, the pellets were digested in 250 μL of 0.25 mg/mL papain solution at 60°C for 24 hours. Once the cell lysate was prepared, the DNA content was quantified using PicoGreen assay kit (Invitrogen) as per manufacturer’s instructions (n=5/group/timepoint). The plate was read at an excitation wavelength of 480 nm and an emission wavelength of 520 nm.

2.3. Trophic Factor Secretion

Single cells were allowed to adhere to tissue culture plastic. Cell culture supernatants were collected from single cell and aggregate groups at days 1, 4, and 7 (n=5/group/timepoint). Cell culture media was changed 24 hours prior to collection. Enzyme-linked immunosorbent assay (ELISA) was used to quantify trophic factors: vascular endothelial growth factor (VEGF), interleukin (IL)-4, and epidermal growth factor (EGF) secreted by monolayers and aggregates. The ELISAs were performed per manufacturer’s (Peprotech) instructions.

2.4. Entrapment of Spheroids in Hydrogels

From the Aggrewell plates, 1200, 2400, and 3600 spheroids (corresponding to 1, 2, or 3 wells at a density of 600 cells/spheroid) were encapsulated in hydrogels (total volume 500 μL) containing fibrinogen (20 mg/mL), thrombin (20 U/mL), calcium chloride (20 mM), protease inhibitor (1:1000), and murine laminin-111 (450 μg/mL) as previously described [26]. After the spheroids were collected from the well plate and centrifuged at 1500 rpm for 5 minutes, the spheroid pellets were resuspended in the fibrinogen solution. The spheroid suspended fibrinogen solution was added to a 48 well plate, and the thrombin, calcium chloride, protease inhibitor, and murine laminin-111 were added and mixed thoroughly. The well plate was incubated at 37 °C for an hour for gelation to occur. The resulting density of spheroids in the hydrogels was 2.4, 4.8, and 7.2 spheroids/μL.

Following gelation, 400 μL of media was placed on top of the 500 μL hydrogel, and the cell culture supernatants were collected on days 1, 4, and 7. Cell culture media was replaced 24 hours prior to collection. VEGF production was measured using an ELISA kit as per manufacturer’s (Peprotech) instructions (n=5/group/timepoint).

2.5. Confocal Microscopy

Prior to spheroid formation, MSCs were stained with Qtracker 525 (Thermo Fisher) following the manufacturer's recommendations. A total of 1200, 2400, and 3600 spheroids were encapsulated in hydrogels as described above. After 24 hours, the hydrogels were imaged using laser scanning confocal microscopy (Leica Confocal SP8, Leica Microsystems Inc.) at 10X magnification.

2.6. Implantation of Therapy into a Murine VML Model

All animal work was performed in accordance with the Animal Welfare Act, the Animal Welfare Regulations, and following the Guide for the Care and Use of Laboratory Animals. The procedures were approved by the Saint Louis University Institutional Animal Care and Use Committee (AUP #2612). Adult (10-12 weeks old) male C57BL6 mice were purchased from Charles Laboratory and housed in an Association for Assessment and Accreditation of Laboratory Animal Care International accredited vivarium. Mice were provided with food and water ad libitum. A total of 33 mice were randomly divided into the different experimental groups. The experimental design is shown in Supp Fig 4. Animals were weighed before surgery and anesthetized with 2.0% isoflurane in oxygen. Sustained release buprenorphine (1 mg/ kg) was administered subcutaneously. The lower hindlimbs were shaved and aseptically prepared. A lateral incision in the skin was made, and blunt dissection separated the musculature from surroundings skin and fascia revealing the gastrocnemius-soleus (GAS) complex. A metal plate was inserted under the GAS complex, and a 3 mm biopsy punch was used to create a full thickness, ~20% muscle mass defect in the belly of the GAS complex [25]. Muscle biopsies were recorded to ensure consistency. Both hindlimbs were injured and received the same treatment.

Qtracker 525 (Thermo Fisher) labeled MSCs were used for spheroid formation 24 hours prior to animal surgery as described above. On the day of animal surgery, spheroids were encapsulated in a hydrogel (with 250 μL of complete media added on top) or resuspended in media in a 1 mL syringe fitted with a 27-gauge needle. Immediately following the encapsulation or resuspension, the spheroids were used in surgery. One group of animals with VML injury received no treatment (NT) and were left untreated (n=4-5/timepoint). The NT group received a 100 μL intramuscular injection of complete media in the medial and lateral gastrocnemius muscles. Another group of mice received a 100 μL intramuscular injection in the medial and lateral gastrocnemius muscles containing 900 Qtracker labeled MSC spheroids (SPH; n=5/timepoint) suspended in complete media. A final subset of animals received treatment consisting of 900 Qtracker labeled MSC spheroids encapsulated in a hydrogel (~125 μL) that was implanted at the defect site (SPH+Gel; n=5/timepoint). Additionally, for the day 14 time-point, a group of mice received VML injury followed by hydrogel treatment (Gel; n=4 mice). Any bleeding was controlled by applying light pressure with a sterile swab, and the wound was closed with interrupted sutures.

Following surgery, animals were placed in fresh cages with water gel and allowed to recover for either 3 or 14 days. At these time-points, mice were euthanized via CO2 asphyxiation. GAS complexes from one leg were cryopreserved for histological analysis and from the other leg were snap frozen for biomolecular analyses

2.7. Histology

Upon collection, the GAS complex was weighed and frozen in 2-methyl butane (ThermoFisher Scientific) that was super-cooled in liquid nitrogen. Muscles were mounted for cryosectioning using optimal cutting temperature (OCT) compound. Cryosections (15 μm) were cut and mounted on slides for analysis. Muscle cross-sections were stained with hematoxylin and eosin (H&E). The muscle cross-sections were also stained for Collagen I (Abcam, ab34710), Myosin heavy chain (DHSB, MF20), F4/80 (ThermoFisher Scientific, PA5-32399), CD31 (R&D Systems, AF3628), CD146 (Proteintech 7564-1-AP), Sca-1 (Abcam Ab 51317), and wheat germ agglutinin (WGA) (W11262) for immunofluorescence analysis. To evaluate MSC distribution in the muscle section using Qtracker, sections were stained with vectashield containing DAPI. For histological staining, sections were fixed in ice-cold acetone or 4% paraformaldehyde, permeabilized using a solution of 0.1% Triton X-100 in phosphate buffered saline (PBS), and blocked in a solution containing 5% Goat Serum, 1% bovine serum albumin (BSA), 0.05% Tween-20. The incubation buffer for both primary and secondary antibodies consisted of 1% BSA and 0.1% Triton X-100 in PBS. All primary antibodies were diluted 1:100, except for CD31, which was diluted at 1:50. CD31 and F4/80 were incubated overnight at 4°C. All other primary antibodies were incubated for 1 hour at 37°C. Appropriate fluorochrome-conjugated secondary antibodies (Invitrogen A-11006, A-11006, A-11078, A-11037, or A2-1141) were diluted at 1:100 and incubated at room temperature for 1 hr. Images were taken using a Zeiss Axiocam microscope at 5× and 20× magnification.

Full-size H&E stained muscle cross-sections were used to quantify the number of myofibers with centrally located nuclei (CLN) at day 14 post-injury (n = 4-5 per group). Muscle sections stained with CD31 at days 14 post-injury were used to quantify CD31 percent (%) area (n = 4/group) via thresholding in FIJI. A total of 2-3 non-overlapping 20× images of defect region from each sample were used for CD31 analysis. The defect region was identified by the high density of nuclei (DAPI+) and low density of myofibers [25]. FITC and DAPI channels were separated and thresholded to measure the percentage of area positively stained CD31. Images of the defect region taken at 5x magnification were used for the quantification of collagen using thresholding in ImageJ [32].

2.8. Myofiber Cross Sectional Area Measurement

Muscle cross-sections from day 14 post-injury were stained with laminin (Abcam) as described above and were scanned to obtain a composite image of the entire muscle (Olympus BX614S). Full-slide scans of the laminin stain were used to determine the myofiber cross-sectional area (CSA, n = 4-5) using a custom-designed image analysis MATLAB program. Briefly, the algorithm thresholds the laminin channel which is then followed by a nonlinear morphological transformation to delineate fiber boundaries and reduce noise. To identify myofibers and avoid spatial variances in brightness, filters for area, circularity, and concavity were applied and a color histogram was computed for each fiber. Fibers were sorted into categories of areas >500, 500-999, 1000–1499, 1500-1999, 2000-2499, 2500-2999, 3000–3499, 3500-3999, 4000–4499, 4500-4999, 5000-5499, 5500-5999, 6000-6499- 6500-7000 and >7000 μm2. The percentage of muscle fibers in each of these categories were determined based on the total number of fibers counted.

2.9. Western Blotting

The harvested muscles were snap frozen in liquid nitrogen and homogenized on ice in RIPA lysis buffer with protease inhibitor cocktail (Sigma) for protein lysate isolation. Protein content was quantified using a Pierce bicinchoninic acid (BCA) Protein Assay Kit (Thermo Scientific). Equal amounts of reduced and denatured protein (60 μg) was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 4–20% precast polyacrylamide gels (Bio-Rad). Protein was transferred onto nitrocellulose membranes with equal protein loading confirmed by Ponceau S staining. Membranes were blocked for 1 hour at room temperature in Tris-buffered saline containing 0.05% (vol/vol) Tween 20 (TBST) and 5% (wt/vol) nonfat dried milk. Membranes were then incubated overnight at 4°C in TBST containing 5% bovine serum albumin (BSA) and primary antibodies diluted 1:1000. Primary antibodies for western blotting included anti-MyoD (Millipore 5.8A Ma141017), anti-myogenin (Millipore MAB3876), anti-a-actinin (Cell Signaling 6487s), anti-desmin (Abcam Ab15200), anti-CD146 (Proteintech 7564-1-AP), Sca-1 (Abcam Ab 51317), anti-heat shock protein-70 (anti-HSP-70; Cell Signaling 4872s), anti-inducible nitric oxide synthase (anti-iNOS; Abcam Ab49999), and anti-arginase (Abcam, Ab91279). After overnight incubation at 4°C, membranes were rinsed three times with TBST for 5 minutes, and then incubated at room temperature for 1 hour in TBST containing 5% nonfat dried milk and the appropriate horseradish peroxidase-conjugated secondary antibody (1:1000, Invitrogen). Appropriate HRP conjugated secondary antibodies (Invitrogen 32260 and 62-6520) were used. Membranes were rinsed three times with TBST for 5 minutes before exposure to ECL reagents (Bio-Rad). The membranes were imaged using the Bio-Rad ChemiDoc (Bio-Rad). Quantification of band intensity was performed using Image J. Data was normalized to Ponceau [33] and used for quantitative analysis.

2.10. Statistical Analysis

Data are presented as a mean ± standard error of the mean. A one-way or two-way analysis of variance was used when appropriate to determine if there was a significant interaction or main effect between variables. When appropriate, a least significant difference (LSD) post-hoc comparison was performed to identify the source of significance with p < 0.05. Statistical analysis between the Gel and the SPH+Gel group was performed using an unpaired two-tailed t-test. Analysis and graphing of data were performed using GraphPad Prism 8 for Windows.

3. Results

3.1. Spheroid size and viability

The viability of MSC in aggregate form was evaluated with live/dead staining (Figure 1A). A Picogreen assay was performed to quantify cell viability and proliferation (Figure 1B). As expected, on day 1, the DNA content was significantly greater in the 600 cells/spheroid group compared to the other groups (p=0.0009). There was no difference in proliferation between the groups at day 4. At day 7, the 300 cells/spheroid had a significantly greater proliferation compared to 150 cell/spheroid (p=0.014). As expected, increasing the number of MSC per spheroid led to a linear increase in the spheroid diameter (Figure 1C). During the 7 days of culture, the spheroids continued to increase in size, but all spheroids were below ~150 μm in diameter.

Figure 1.

Figure 1.

Open in a new tab

Spheroids formed using 150, 300, and 500 cells were analyzed for size and cellular viability. (A) Live/dead imaging confirmed the maintained cell viability in spheroid form over 7 days in culture. (B) Picogreen assay indicated a linear increase in DNA content in all spheroids from days 1-7 (D1-D7). (C) Spheroid diameter increased linearly with increasing number of cells. The diameter of spheroids also increased over time during culture. Quantification of (D) VEGF, (E) EGF, and (F) IL-4 released from murine mesenchymal stem cell spheroids on days 1, 4, and 7 of culture. The red dashed lines indicate the concentration value of single cell control. Symbols for statistical differences between D1-D4 is (α), D1-D7 is (Φ), and D4-D7 is (λ), and “*” indicates a significant difference between treatment groups is (p<0.05).

3.2. Trophic Factor Secretion

The production of VEGF from MSC spheroids increased linearly with the number of MSCs used for aggregation (Figure 1D). At days 1,4, and 7 of culture, a significantly greater amount of VEGF was released from 600 cells/spheroid released compared to 300 cells/spheroid and 150 cells/spheroid. In the 600 cell/spheroid group, there was a significantly greater amount of VEGF released at day 4 compared to day 1 (p=0.0191) and at day 7 compared to day 4 (p=0.0001). Surprisingly, all spheroids released a lower amount of VEGF compared to single cell controls. EGF production from spheroids showed no differences between groups but its concentration was comparable to the single cell control (Figure 1E). The production of IL-4 was significantly higher from the 600 cells/spheroid group on days 4 and 7 (p=0.0001) (Figure 1F). The concentration of IL-4 released from the 600 cells/spheroid group was well above that of single cell controls on days 4 and 7.

3.3. MSC spheroid encapsulation in hydrogels

Qtracker labeled MSCs in aggregate form were encapsulated at densities of 1200, 2400, 3600 spheroids in LM-111 enriched fibrin hydrogels (Figure 2A). Confocal imaging of the hydrogels 24 hours post-encapsulation indicated that the spheroids maintained a spherical morphology and were evenly distributed throughout the gels. The production of VEGF from hydrogel spheroids increased linearly with time (Figure 2B). On day 4, the production of VEGF in the 3600 spheroids/gel group was significantly higher compared to 1200 and 2400 spheroids/gel groups. At day 7, the production of VEGF was significantly greater in the 2400 and 3600 spheroids/gel groups compared to the 1200 spheroids/gel group. These results confirmed that MSCs remain functional when encapsulated at a density of 3600 spheroids in the hydrogels. Due to the low concentration of IL-4 released from spheroids (Fig. 1F) and no differences in EGF release (Fig. 1E), we chose to measure only VEGF release from the spheroids encapsulated in hydrogels.

Figure 2.

Figure 2.

Open in a new tab

MSC spheroids were encapsulated at a density of 1200, 2400, and 3600 spheroids/hydrogel. (A) Qtracker labeled MSC spheroid morphology 24 hours after encapsulated in fibrin-laminin hydrogel (10x magnification). Scale Bars are 200 μm. (B) Quantification of VEGF released from hydrogel encapsulated MSC spheroids on days 1, 4, and 7 of culture. “*” indicates a significant difference between treatment groups is (p<0.05). Symbols for statistical differences between D1-D4 is (α), D1-D7 is (Φ), and D4-D7 is (λ), and “*” indicates a significant difference between treatment groups is (p<0.05).

3.4. Cell distribution in VML injured muscles

VML injured GAS muscles received no treatment (NT) or were treated with Qtracker labeled MSC spheroids (SPH) or Qtracker labeled MSC spheroids encapsulated in a hydrogel (SPH+Gel). No statistical differences were noted in the biopsy punch weight between the various treatment groups. The presence of MSCs in the injured muscles was assessed qualitatively by imaging Qtracker stained cells co-localizing with DAPI on day 3 post-injury (Figure 3). Qtracker staining could not be detected in the muscle cross-sections at day 14 post-injury. H&E stained images of the same sample from a similar region of the muscle are displayed for comparison. In the NT VML injured muscles, some acellular structures displayed autofluorescence in the FITC channel but did not co-localize with DAPI. In the spheroid injection group, Qtracker+ DAPI+ cells were found clustered in the defect region. In the spheroid + gel group, the remodeled hydrogel could be identified in the defect. Qtracker+ DAPI+ MSC spheroids could be detected within the pore network of the gel.

Figure 3.

Figure 3.

Open in a new tab

The presence of MSCs in the defect at 3 days post-injury. The white arrows point to Qtracker labeled MSCs co-localizing with DAPI. The orange arrows point to auto fluorescing structures that did not co-localize with DAPI. In the H&E images, the black arrows point to cellular aggregates in the pore network of the gel and the dotted line marks the boundary of the remodeled hydrogel from the surrounding muscle. NT, No Treatment; SPH, injected spheroids; SPH+Gel, spheroids encapsulated in gel. All pictures were taken at 20X. Scale bars are 100 μm.

3.5. Cellular infiltration in VML injured muscles

The presence of CD146+ cells on day 3 post-injury in the VML defect (Figure 4A) as well as the remaining muscle mass (Supp Fig. 1B) are shown. Qualitative analysis of the images suggests more CD146+ cells in the defect in the treated groups compared to the NT group. Quantification via western blotting suggests that the amount of CD146+ was significantly greater in both the SPH as well as the SPH+Gel treated groups compared to the NT group (Figure 4D).

Figure 4.

Figure 4.

Open in a new tab

VML injured muscles were stained for (A) CD146+ cells at 3 days post injury (B) endothelial cells (CD31+) as well as (C) myosin heavy chain (MHC) and collagen (COL) at day 14 post-injury (n=4-5/group). Treated muscles showed increased presence of (D) CD146 by western blotting (E) CD31+ endothelial cell infiltration, and reduced (F) collagen deposition compared to muscles that received no treatment (NT). SPH=injected spheroids; SPH+Gel=spheroids encapsulated in gel. All pictures were taken at 20X. Scale bars are 100 μm. Scale Bars are 200 μm in MHC/COL/DAPI. “*” indicates a significant difference between treatment groups is (p<0.05).

Muscle cross-sections were also stained with SCA-1 on day 3 post-injury (Supp Fig. 2A). Qualitative analysis of the image shows that there was more SCA-1+ cells in the interstitial space around the myofibers in the remaining muscle mass compared to the defect in all groups. Quantification of SCA-1 protein via western blotting showed no significant difference between the groups (Supp Fig. 2B).

CD31 staining was performed on muscle tissue sections to assess endothelial cell infiltration and angiogenic activity on day 14 post-injury (Figure 4B). All VML injured groups supported CD31+ cell infiltration in the defect at day 14 post-injury. More mature vessel-like structures with a visible lumen could be identified in the defect in treated groups whereas the NT group mainly showed punctate staining of CD31+ individual endothelial cells. Quantitative analysis of the defect region at day 14 showed a significant increase in CD31+ cell infiltration in the spheroid as well as the SPH+Gel group compared to the NT group (Figure 4E).

3.6. Muscle Regeneration and Morphology

Muscle cross-sections stained with MHC and collagen on day 14 post-injury are shown in Figure 4C. The untreated muscles showed a large defect region filled with collagenous fibrotic tissue. However, in both treated groups, significantly less COL+ fibrotic tissue was present (Figure 4F). In both treated groups, there was a greater number of MHC+ myofibers in and around the defect region compared to the untreated group.

Increased cellular infiltration was observed in all groups but the hydrogel could not be identified in the H&E images on day 14 suggesting that it had undergone complete remodeling by this time-point (Figure 5A). On day 3 post-injury, a significant increase in muscle mass was observed in the SPH+Gel group compared to the SPH alone group (Figure 5B). However, this increase in muscle mass could be attributed to the weight of the implanted gel. On day 14, there was an 8.2% increase GAS muscle mass when normalized to body weight in the SPH+Gel group compared to the NT group (p=0.0326), and a 9.81% increase in normalized muscle mass was observed in the SPH+Gel group compared to the SPH injection group (p=0.0089). On day 14, quantitative analysis revealed that there was a significant increase in myofibers with CLN in the SPH+Gel group compared to the NT group and a trend towards an increase (p=0.0739) between the spheroid injection and the SPH+Gel group, indicative of enhanced myofiber regeneration (Figure 5C). The hydrogel could be identified in the Gel group at day 14 (Supp Fig. 3A) but no differences were noted in the muscle mass or myofibers with CLN between the Gel and SPH+Gel group (Supp Fig. 3 B-C).

Figure 5.

Figure 5.

Open in a new tab

VML injured muscles were stained with (A) H&E at day 14 post-injury. Scale Bars are 100 μm. (B) SPH+Gel group showed higher muscle mass at day 14 compared to other groups (n=4-5/group). A higher quantity of myofibers with (C) centrally located nuclei (CLN) were quantified (n = 4-5/group), and (D) small cross-sectional area (CSA) (<500 μm2) were present in SPH+Gel treated muscles at day 14 (n=4-5/group). (E) Laminin-stained muscle sections used for CSA quantification are shown. “*” indicates a significant difference between treatment groups is (p<0.05).

3.7. Myofiber Cross-Sectional Area

Transverse cross-sections of GAS muscles were stained with laminin and are shown in Figure 5E. The distribution of fiber CSA is seen in Figure 5D. The SPH+Gel treatment significantly increased the percentage of small size myofibers (<500 μm2) compared to the NT and SPH alone groups. The percentage of fibers in the 500-999 μm2 was significantly higher in the NT group compared to the SPH+Gel group (p=0.0119). The mean CSA was significantly greater in the cage control compared to NT group (p=0.0110) and spheroids + gel group (p=0.0234) (Supp Fig. 2C). The total number of myofibers in the cage control GAS muscles was determined to be ~ 6118. On day 14, VML injury reduced the number of myofibers in the GAS complex by ~ 41% (Supp Fig. 2D). In support, a previous study also reported that VML injury (~20% defect) reduced the number of muscle fibers by ~40% at 8 weeks post-injury in a rat model [34].

3.8. Myogenic Protein Expression

Muscle lysates were collected at both days 3 and 14 and probed with myogenic markers MyoD, myogenin, desmin, and alpha-actinin (Figure 6A). No significant differences between groups were observed on day 3. On day 14, the expression of both MyoD and Myogenin was significantly higher in the SPH+Gel group compared to both NT and SPH injection groups (Figure 6B, C). Compared to the SPH injection group, the SPH+Gel group showed a ~3.6 fold increase in MyoD expression and a ~6 fold increase in myogenin expression. The expression of desmin and alpha-actinin showed no differences between the groups on day 14 (Figure 6D, E). The Gel group showed higher expression of MyoD, Arginase, and HSP-70 compared to the SPH+Gel group (Supp Fig. 3D, I, and J respectively). The expression of myogenin, desmin, alpha-actinin, and iNOS showed no statistical differences between the SPH+Gel and the Gel group (Supp Fig. 3E-H respectively).

Figure 6.

Figure 6.

Open in a new tab

Muscle protein lysates were probed for myogenic and immunogenic marker protein expression (n=4-5/group). (A) Representative bands for each protein are shown. The quantification of (B) MyoD, (C) Myogenin, (D) Desmin, and (E) α-Actinin, (F) iNOS, (G) Arginase, and (H) HSP-70 are shown. “*” indicates a significant difference between treatment groups is (p<0.05).

3.9. Immune Response

Macrophage (F4/80+) infiltration was increased on day 14 post-VML injury in all groups, irrespective of treatment (Supp Fig. 1C). Muscle lysates were also probed for macrophage phenotype markers [35, 36] such as inducible nitric oxide synthase (iNOS), and arginase, as well as heat shock protein (HSP-70) (Figure 6A). No significant differences between groups were observed on day 3. On day 14, iNOS, a pro-inflammatory M1-like phenotype marker was significantly higher in the SPH+Gel group compared to both NT and SPH injection group at day 14 (Figure 6F). The expression of iNOS was also found significantly lower in the NT and SPH injection groups at day 3 compared to day 14. However, its expression was maintained over time in the SPH+Gel group. Arginase, an anti-inflammatory M2 macrophage marker, was significantly higher in the SPH+Gel group compared to both NT and SPH injection group on day 14 (Figure 6G). Similar to iNOS, arginase expression decreased in the NT and SPH injection groups over time but was maintained in the SPH+Gel group. There were no differences in HSP-70, a marker indicative of cellular stress, between the groups at both days 3 and 14 (Figure 6H).

4. Discussion

In this study, MSC spheroids co-delivered with a fibrin-laminin hydrogel improved skeletal muscle regeneration to a greater extent than MSC spheroids injected directly into the injured muscles. Both the injected SPH and SPH+Gel groups showed similar improvements in the vascular niche, as indicated by the increased presence of CD146+ pericytes on day 3 and CD31+ endothelial cells or vessel-like structures on day 14. Both SPH and SPH+Gel groups also reduced fibrotic tissue deposition in the VML injured muscles. In support, previous studies have shown improved angiogenesis [37] and reduced collagen deposition [38] following MSC transplantation in skeletal muscle. However, improvements in muscle mass, myogenesis, and regenerating myofibers were only observed in the SPH+Gel group.

We could not reliably assess the co-localization of Qtracker+ and CD146+ cells to distinguish between resident and transplanted cells. However, an overall increase in CD146+ pericyte presence in the VML defect was found in both treatment groups. While CD146+ pericytes exhibit myogenic potential [39] and can increase IGF-1 signaling [40], an increase in pericyte presence was not sufficient to improve myofiber regeneration following VML. Similarly, anincrease in CD31+ cells/vessels indicative of angiogenesis by both treatment groups did not translate into improved skeletal muscle regeneration.

Based on previous studies [22-24], it is reasonable to assume that the hydrogel may have offered protection to the entrapped spheroids from the immune cells and cytotoxic cytokines present in the VML wound site. It is possible that the MSC spheroids delivered via the hydrogel persisted in larger numbers and for a longer period post-transplantation than the spheroids injected directly into the muscles. The remodeled hydrogel could be identified in the muscle cross-sections at day 3 but not at day 14 post-injury. Therefore, the hydrogel likely underwent complete remodeling between days 3-14, thereby releasing the spheroids in the VML microenvironment. In support of this contention, iNOS and Arginase expression decreased in the SPH group by day 14, suggesting downregulation of the immune response. However, their expression was maintained over time in the SPH+Gel group suggesting ongoing macrophage mediated immune response to the transplanted therapy. Inducible nitric oxide synthase (iNOS) and arginase are enzymes that are widely used as the markers for macrophage phenotype (M1/M2) characterization [35, 36]. iNOS metabolizes the amino acid arginine to produce nitric oxide which inhibits cell proliferation while arginase metabolizes Arginine to produce ornithine which boosts cell proliferation and initiates tissue repair via the production of polyamines and collagen. Some studies have also suggested a dual role for the M1 macrophages in muscle injuries where they are beneficial in reducing fibrosis [41]. Therefore, the persistent presence of M1 macrophages may not be completely detrimental to regeneration.

It has been shown that trophic factor secretion is the primary mechanism of repair by MSCs. In this study, MSC spheroids showed a linear increase in VEGF and IL-4 secretion with increasing cell number used for aggregation. VEGF can promote satellite cell proliferation and migration and exert anti-apoptotic effects on myoblasts [26, 42, 43]. IL-4 is an anti-inflammatory marker, has been seen to promote myoblast differentiation and fusion into myotubes [44, 45]. EGF can promote satellite cell proliferation and differentiation [46, 47] but its secretion was found unmodulated by cell number.

To determine the extent of myogenic protein expression, we quantified markers associated with satellite cell proliferation and differentiation. MyoD is one of the earliest markers of myogenic commitment and marks myoblast proliferation while myogenin is a marker of myoblast terminal differentiation. At day 14, MyoD and Myogenin expression was significantly higher in the SPH+Gel group suggesting greater myoblast proliferation and differentiation than the SPH group.

To further assess myofiber regeneration, we quantified the number of myofibers with CLN. At 14 days- post- injury, the presence of myofibers with CLN primarily indicates myofiber regeneration. The SPH+Gel group had a significantly greater number of CLN compared to NT group. Fiber size distribution shows an increase in the percentage of small diameter fibers (<500 μm2) indicative of regenerating myofibers at 14 days-post-injury in the SPH+Gel group compared to the other groups. Finally, the normalized muscle mass also showed a small but significant increase in the SPh+Gel group at 14-days-post-injury compared to the other groups. Taken together, a modest improvement in muscle mass (8.2% compared to untreated and 9.8% compared to the SPH group), increased presence of small and nucleated myofibers, and elevated myogenic protein expression would indicate a higher regenerative response post-VML in the SPH+Gel group.

A limitation of this study is the lack of a hydrogel only control group. We havecompared the SPH+Gel group with the hydrogel alone (Gel) group at 14 days post-VML injury (Supp Fig. 3). While we did not observe statistical differences in muscle mass and myofibers with CLN, the Gel group showed higher expression of MyoD, Arginase, and HSP-70 compared to the SPH+Gel group. These results could be explained in part by the slower degradation of the hydrogel in the absence of encapsulated spheroids. In support, the hydrogel could still be identified in the histological cross-sections at 14 days post-injury in the Gel group but was completely remodeled in the SPH+Gel group and could not be identified histologically by the 14 day time-point. It can also be speculated that the MSC spheroids released after hydrogel remodeling in the SPH+Gel group elicited immunomodulation which reduced the expression of Arginase and HSP-70 and suppressed the stimulation of satellite cells into cell cycle thereby reducing the expression of MyoD.

It is likely that the MSC spheroids and the fibrin-laminin hydrogel acted synergistically to promote skeletal muscle regeneration following VML. In this study, the specific contribution of spheroids and the hydrogel towards muscle regeneration in the combined (SPH+Gel) treatment group was not determined. Additional studies are needed to understand the interaction between MSC spheroids and ECM components such as fibrin and LM. In future studies, extended time-points (4-6 weeks) will be evaluated to determine the extent of functional recovery and myofiber innervation. Another limitation of this study is the small animal model and size of the VML defect. Future studies should investigate the efficacy of these therapies in clinically relevant animal models (e.g., porcine) with large VML defects. The dimensions of the hydrogel can be easily altered to fit the defect size. The number of spheroids used for transplantation can also be increased proportionally. Some studies have also explored the use of spinner flasks and bioreactors for large scale generation of spheroids [48].

In conclusion, hydrogel encapsulation of MSC spheroids leads to better regenerative outcomes compared to direct injection of MSC spheroids in the injured muscle. The direct injection of MSC spheroids enhanced angiogenesis and reduced fibrosis in the VML injured muscle but improvements in muscle mass, myogenesis, and regenerating myofibers were not observed.

Supplementary Material

fS2

Supplemental Figure 2. The presence of (A) SCA-1+ cells observed in the muscle 3 days post-injury in the defect and in the interstitial space. (B) Representative bands and quantification of SCA-1 are shown. All pictures were taken at 20X. VML injury reduced the (C) mean CSA and the (D) total number of myofibers. NT, No Treatment; SPH, injected spheroids; SPH+Gel, spheroids encapsulated in gel. Scale bars are 100 μm. “*” indicates a significant difference between treatment groups is (p<0.05).

fS1

Supplemental Figure 1. (A) MSCs express both CD146+ and SCA-1+ in vitro. (B) VML injured muscles were stained for CD146+ cells at 3 days post injury and in the interstitial space around the myofibers (n=4-5/group). (C) The presence of macrophages (F4/80+) was observed in the defect region post-VML injury (n=4-5/group). All pictures were taken at 20X. NT, No Treatment; SPH, injected spheroids; SPH+Gel, spheroids encapsulated in gel. Scale bars are 100 μm.

fS4

Supplemental Figure 4. A schematic depicting the experimental design of the study.

fS3

Supplemental Figure 3. (A) Muscle cross-section showing the presence of the hydrogel (outlined using black broken lines) in the Gel group at day 14 post-VML injury. A comparison of (B) muscle weights, and (C) myofibers with CLN between the SPH+Gel and the Gel group shows no statistical differences. The protein expression of markers was normalized to NT group. Fold change over NT group is presented for (D) MyoD, (E) Myogenin, (F) alpha-actinin, (G) desmin, (H) iNOS, (I) arginase, (J) HSP-70. “*” indicates a significant difference between treatment groups is (p<0.05).

Acknowledgements:

The authors would like to thank Caroline Murphy and Dr. Grant Kolar (Saint Louis University) for technical assistance with histological imaging.

Funding:

This work was supported by a grant from the Department of Defense (Applied Research Award -PRORP) OR170286 as well as National Institute of Health (NIGMS) 1R15GM129731, both awarded to KG.

Footnotes

Conflict of Interest: The authors have no conflicts of interest to declare.

Ethics Statement: The authors confirm that the ethical policies of the journal, as noted on the journal’s author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received.

References

  • 1.Corona BT, Rivera JC, Owens JG, Wenke JC, and Rathbone CR, Volumetric muscle loss leads to permanent disability following extremity trauma. J Rehabil Res Dev, 2015. 52(7): p. 785–92. [DOI] [PubMed] [Google Scholar]
  • 2.Larouche J, Greising SM, Corona BT, and Aguilar CA, Robust inflammatory and fibrotic signaling following volumetric muscle loss: a barrier to muscle regeneration. Cell Death Dis, 2018. 9(3): p. 409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Boppart MD, De Lisio M, Zou K, and Huntsman HD, Defining a role for non-satellite stem cells in the regulation of muscle repair following exercise. Front Physiol, 2013. 4: p. 310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Krueger TEG, Thorek DLJ, Denmeade SR, Isaacs JT, and Brennen WN, Concise Review: Mesenchymal Stem Cell-Based Drug Delivery: The Good, the Bad, the Ugly, and the Promise. Stem Cells Transl Med, 2018. 7(9): p. 651–663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Galipeau J, The mesenchymal stromal cells dilemma--does a negative phase III trial of random donor mesenchymal stromal cells in steroid-resistant graft-versus-host disease represent a death knell or a bump in the road? Cytotherapy, 2013. 15(1): p. 2–8. [DOI] [PubMed] [Google Scholar]
  • 6.Tongers J, Losordo DW, and Landmesser U, Stem and progenitor cell-based therapy in ischaemic heart disease: promise, uncertainties, and challenges. Eur Heart J, 2011. 32(10): p. 1197–206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Lee S, Choi E, Cha MJ, and Hwang KC, Cell adhesion and long-term survival of transplanted mesenchymal stem cells: a prerequisite for cell therapy. Oxid Med Cell Longev, 2015. 2015: p. 632902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Li L, Chen X, Wang WE, and Zeng C, How to Improve the Survival of Transplanted Mesenchymal Stem Cell in Ischemic Heart? Stem Cells Int, 2016. 2016: p. 9682757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Burdick JA, Mauck RL, and Gerecht S, To Serve and Protect: Hydrogels to Improve Stem Cell-Based Therapies. Cell Stem Cell, 2016. 18(1): p. 13–5. [DOI] [PubMed] [Google Scholar]
  • 10.McGuigan AP, Bruzewicz DA, Glavan A, Butte MJ, and Whitesides GM, Cell encapsulation in sub-mm sized gel modules using replica molding. PLoS One, 2008. 3(5): p. e2258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Cesarz Z and Tamama K, Spheroid Culture of Mesenchymal Stem Cells. Stem Cells Int, 2016. 2016: p. 9176357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Sart S, Tsai AC, Li Y, and Ma T, Three-dimensional aggregates of mesenchymal stem cells: cellular mechanisms, biological properties, and applications. Tissue Eng Part B Rev, 2014. 20(5): p. 365–80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Langenbach F, Berr K, Naujoks C, Hassel A, Hentschel M, Depprich R, Kubler NR, Meyer U, Wiesmann HP, Kogler G, and Handschel J, Generation and differentiation of microtissues from multipotent precursor cells for use in tissue engineering. Nat Protoc, 2011. 6(11): p. 1726–35. [DOI] [PubMed] [Google Scholar]
  • 14.Baraniak PR and McDevitt TC, Scaffold-free culture of mesenchymal stem cell spheroids in suspension preserves multilineage potential. Cell Tissue Res, 2012. 347(3): p. 701–11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Bhang SH, Lee S, Shin JY, Lee TJ, and Kim BS, Transplantation of cord blood mesenchymal stem cells as spheroids enhances vascularization. Tissue Eng Part A, 2012. 18(19–20): p. 2138–47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Lee JH, Han YS, and Lee SH, Long-Duration Three-Dimensional Spheroid Culture Promotes Angiogenic Activities of Adipose-Derived Mesenchymal Stem Cells. Biomol Ther (Seoul), 2016. 24(3): p. 260–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Ruehle MA, Stevens HY, Beedle AM, Guldberg RE, and Call JA, Aggregate mesenchymal stem cell delivery ameliorates the regenerative niche for muscle repair. J Tissue Eng Regen Med, 2018. 12(8): p. 1867–1876. [DOI] [PubMed] [Google Scholar]
  • 18.Amer MH, Rose F, Shakesheff KM, and White LJ, A biomaterials approach to influence stem cell fate in injectable cell-based therapies. Stem Cell Res Ther, 2018. 9(1): p. 39. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Falanga V, Iwamoto S, Chartier M, Yufit T, Butmarc J, Kouttab N, Shrayer D, and Carson P, Autologous bone marrow-derived cultured mesenchymal stem cells delivered in a fibrin spray accelerate healing in murine and human cutaneous wounds. Tissue Eng, 2007. 13(6): p. 1299–312. [DOI] [PubMed] [Google Scholar]
  • 20.Sorrell JM and Caplan AI, Topical delivery of mesenchymal stem cells and their function in wounds. Stem Cell Res Ther, 2010. 1(4): p. 30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Madl CM and Heilshorn SC, Engineering Hydrogel Microenvironments to Recapitulate the Stem Cell Niche. Annu Rev Biomed Eng, 2018. 20: p. 21–47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.He J, Zhang N, Zhu Y, Jin R, and Wu F, MSC spheroids-loaded collagen hydrogels simultaneously promote neuronal differentiation and suppress inflammatory reaction through PI3K-Akt signaling pathway. Biomaterials, 2021. 265: p. 120448. [DOI] [PubMed] [Google Scholar]
  • 23.Murphy KC, Fang SY, and Leach JK, Human mesenchymal stem cell spheroids in fibrin hydrogels exhibit improved cell survival and potential for bone healing. Cell Tissue Res, 2014. 357(1): p. 91–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Ho SS, Murphy KC, Binder BY, Vissers CB, and Leach JK, Increased Survival and Function of Mesenchymal Stem Cell Spheroids Entrapped in Instructive Alginate Hydrogels. Stem Cells Transl Med, 2016. 5(6): p. 773–81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Marcinczyk M, Dunn A, Haas G, Madsen J, Scheidt R, Patel K, Talovic M, and Garg K, The Effect of Laminin-111 Hydrogels on Muscle Regeneration in a Murine Model of Injury. Tissue Eng Part A, 2019. 25(13–14): p. 1001–1012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Marcinczyk M, Elmashhady H, Talovic M, Dunn A, Bugis F, and Garg K, Laminin-111 enriched fibrin hydrogels for skeletal muscle regeneration. Biomaterials, 2017. 141: p. 233–242. [DOI] [PubMed] [Google Scholar]
  • 27.Zou K, De Lisio M, Huntsman HD, Pincu Y, Mahmassani Z, Miller M, Olatunbosun D, Jensen T, and Boppart MD, Laminin-111 improves skeletal muscle stem cell quantity and function following eccentric exercise. Stem Cells Transl Med, 2014. 3(9): p. 1013–22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Peng KY, Liu YH, Li YW, Yen BL, and Yen ML, Extracellular matrix protein laminin enhances mesenchymal stem cell (MSC) paracrine function through alphavbeta3/CD61 integrin to reduce cardiomyocyte apoptosis. J Cell Mol Med, 2017. 21(8): p. 1572–1583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Valero MC, Huntsman HD, Liu J, Zou K, and Boppart MD, Eccentric exercise facilitates mesenchymal stem cell appearance in skeletal muscle. PLoS One, 2012. 7(1): p. e29760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Talovic M, Patel K, Schwartz M, Madsen J, and Garg K, Decellularized extracellular matrix gelloids support mesenchymal stem cell growth and function in vitro. Journal of Tissue Engineering and Regenerative Medicine, 2019. 13(10): p. 1830–1842. [DOI] [PubMed] [Google Scholar]
  • 31.Feng J, Mineda K, Wu SH, Mashiko T, Doi K, Kuno S, Kinoshita K, Kanayama K, Asahi R, Sunaga A, and Yoshimura K, An injectable non-cross-linked hyaluronic-acid gel containing therapeutic spheroids of human adipose-derived stem cells. Sci Rep, 2017. 7(1): p. 1548. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Garg K, Ward CL, Rathbone CR, and Corona BT, Transplantation of devitalized muscle scaffolds is insufficient for appreciable de novo muscle fiber regeneration after volumetric muscle loss injury. Cell Tissue Res, 2014. 358(3): p. 857–73. [DOI] [PubMed] [Google Scholar]
  • 33.Fosang AJ and Colbran RJ, Transparency Is the Key to Quality. The Journal of biological chemistry, 2015. 290(50): p. 29692–29694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Ward CL, Ji L, and Corona BT, An Autologous Muscle Tissue Expansion Approach for the Treatment of Volumetric Muscle Loss. Biores Open Access, 2015. 4(1): p. 198–208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Yang Z and Ming XF, Functions of arginase isoforms in macrophage inflammatory responses: impact on cardiovascular diseases and metabolic disorders. Front Immunol, 2014. 5: p. 533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Mills CD, M1 and M2 Macrophages: Oracles of Health and Disease. Crit Rev Immunol, 2012. 32(6): p. 463–88. [DOI] [PubMed] [Google Scholar]
  • 37.Huntsman HD, Zachwieja N, Zou K, Ripchik P, Valero MC, De Lisio M, and Boppart MD, Mesenchymal stem cells contribute to vascular growth in skeletal muscle in response to eccentric exercise. Am J Physiol Heart Circ Physiol, 2013. 304(1): p. H72–81. [DOI] [PubMed] [Google Scholar]
  • 38.Qiu X, Liu S, Zhang H, Zhu B, Su Y, Zheng C, Tian R, Wang M, Kuang H, Zhao X, and Jin Y, Mesenchymal stem cells and extracellular matrix scaffold promote muscle regeneration by synergistically regulating macrophage polarization toward the M2 phenotype. Stem Cell Res Ther, 2018. 9(1): p. 88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Klimczak A, Kozlowska U, and Kurpisz M, Muscle Stem/Progenitor Cells and Mesenchymal Stem Cells of Bone Marrow Origin for Skeletal Muscle Regeneration in Muscular Dystrophies. Archivum immunologiae et therapiae experimentalis, 2018. 66(5): p. 341–354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Brun CE, Chevalier FP, Dumont NA, and Rudnicki MA, Chapter 10 - The Satellite Cell Niche in Skeletal Muscle, in Biology and Engineering of Stem Cell Niches, Vishwakarma A and Karp JM, Editors. 2017, Academic Press: Boston. p. 145–166. [Google Scholar]
  • 41.Novak ML, Weinheimer-Haus EM, and Koh TJ, Macrophage activation and skeletal muscle healing following traumatic injury. The Journal of pathology, 2014. 232(3): p. 344–355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Ojima K, Oe M, Nakajima I, Shibata M, Chikuni K, Muroya S, and Nishimura T, Proteomic analysis of secreted proteins from skeletal muscle cells during differentiation. EuPA Open Proteomics, 2014. 5: p. 1–9. [Google Scholar]
  • 43.Germani A, Di Carlo A, Mangoni A, Straino S, Giacinti C, Turrini P, Biglioli P, and Capogrossi MC, Vascular Endothelial Growth Factor Modulates Skeletal Myoblast Function. The American Journal of Pathology, 2003. 163(4): p. 1417–1428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Tidball JG and Villalta SA, Regulatory interactions between muscle and the immune system during muscle regeneration. American journal of physiology. Regulatory, integrative and comparative physiology, 2010. 298(5): p. R1173–R1187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Tidball JG and Wehling-Henricks M, Macrophages promote muscle membrane repair and muscle fibre growth and regeneration during modified muscle loading in mice in vivo. The Journal of physiology, 2007. 578(Pt 1): p. 327–336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Feige P, Tsai EC, and Rudnicki MA, Analysis of human satellite cell dynamics on cultured adult skeletal muscle myofibers. Skeletal Muscle, 2021. 11(1): p. 1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Zhou S.a., Zhang W, Cai G, Ding Y, Wei C, Li S, Yang Y, Qin J, Liu D, Zhang H, Shao X, Wang J, Wang H, Yang W, Wang H, Chen S, Hu P, and Sun L, Myofiber necroptosis promotes muscle stem cell proliferation via releasing Tenascin-C during regeneration. Cell Research, 2020. 30(12): p. 1063–1077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Massai D, Isu G, Madeddu D, Cerino G, Falco A, Frati C, Gallo D, Deriu MA, Falvo D'Urso Labate G, Quaini F, Audenino A, and Morbiducci U, A Versatile Bioreactor for Dynamic Suspension Cell Culture. Application to the Culture of Cancer Cell Spheroids. PLoS One, 2016. 11(5): p. e0154610. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

fS2

Supplemental Figure 2. The presence of (A) SCA-1+ cells observed in the muscle 3 days post-injury in the defect and in the interstitial space. (B) Representative bands and quantification of SCA-1 are shown. All pictures were taken at 20X. VML injury reduced the (C) mean CSA and the (D) total number of myofibers. NT, No Treatment; SPH, injected spheroids; SPH+Gel, spheroids encapsulated in gel. Scale bars are 100 μm. “*” indicates a significant difference between treatment groups is (p<0.05).

NIHMS1742871-supplement-fS2.tif (1.1MB, tif)
fS1

Supplemental Figure 1. (A) MSCs express both CD146+ and SCA-1+ in vitro. (B) VML injured muscles were stained for CD146+ cells at 3 days post injury and in the interstitial space around the myofibers (n=4-5/group). (C) The presence of macrophages (F4/80+) was observed in the defect region post-VML injury (n=4-5/group). All pictures were taken at 20X. NT, No Treatment; SPH, injected spheroids; SPH+Gel, spheroids encapsulated in gel. Scale bars are 100 μm.

NIHMS1742871-supplement-fS1.tif (2.2MB, tif)
fS4

Supplemental Figure 4. A schematic depicting the experimental design of the study.

NIHMS1742871-supplement-fS4.tif (278.3KB, tif)
fS3

Supplemental Figure 3. (A) Muscle cross-section showing the presence of the hydrogel (outlined using black broken lines) in the Gel group at day 14 post-VML injury. A comparison of (B) muscle weights, and (C) myofibers with CLN between the SPH+Gel and the Gel group shows no statistical differences. The protein expression of markers was normalized to NT group. Fold change over NT group is presented for (D) MyoD, (E) Myogenin, (F) alpha-actinin, (G) desmin, (H) iNOS, (I) arginase, (J) HSP-70. “*” indicates a significant difference between treatment groups is (p<0.05).

NIHMS1742871-supplement-fS3.tif (2.3MB, tif)

RESOURCES