Adipose-Derived Stem Cell Sheets Improve Early Biomechanical Graft Strength in Rabbits After Anterior Cruciate Ligament Reconstruction

Abstract

Background:

Although various reconstruction techniques are available for anterior cruciate ligament (ACL) injuries, a long recovery time is required before patients return to sports activities, as the reconstructed ACL requires time to regain strength. To date, several studies have reported use of mesenchymal stem cells in orthopaedic surgery; however, no studies have used adipose-derived stem cell (ADSC) sheets in ACL reconstruction (ACLR).

Hypothesis:

ADSC sheet transplantation can improve biomechanical strength of the autograft used in ACLR.

Study Design:

Controlled laboratory study.

Methods:

A total of 68 healthy Japanese white rabbits underwent unilateral ACLR with a semitendinosus tendon autograft after random enrollment into a control group (no sheet; n = 34) and a sheet group (ADSC sheet; n = 34). At 2, 4, 8, 16, and 24 weeks after surgery, rabbits in each group were sacrificed to evaluate tendon-bone healing using histological staining, micro–computed tomography, and biomechanical testing. At 24 weeks, scanning transmission electron microscopy of the graft midsubstance was performed.

Results:

The ultimate failure load for the control and sheet groups, respectively, was as follows: 17.2 ± 5.5 versus 37.3 ± 10.3 (P = .01) at 2 weeks, 28.6 ± 1.9 versus 47.4 ± 10.4 (P = .003) at 4 weeks, 53.0 ± 14.3 versus 48.1 ± 9.3 (P = .59) at 8 weeks, 66.2 ± 9.3 versus 95.2 ± 43.1 (P = .24) at 16 weeks, and 66.7 ± 27.3 versus 85.3 ± 29.5 (P = .39) at 24 weeks. The histological score was also significantly higher in the sheet group compared with the control group at early stages up to 8 weeks. On micro–computed tomography, relative to the control group, the bone tunnel area was significantly narrower in the sheet group at 4 weeks, and the bone volume/tissue volume of the tendon-bone interface was significantly greater at 24 weeks. Scanning transmission electron microscopy at 24 weeks indicated that the mean collagen fiber diameter in the midsubstance was significantly greater, as was the occupation ratio of collagen fibers per field of view, in the sheet group.

Conclusion:

ADSC sheets improved biomechanical strength, prevented bone tunnel enlargement, and promoted tendon-bone interface healing and graft midsubstance healing in an in vivo rabbit model.

Clinical Relevance:

ADSC sheets may be useful for early tendon-bone healing and graft maturation in ACLR.

Keywords

anterior cruciate ligament semitendinosus tendon tendon-bone healing adipose-derived stem cell cell sheet

An anterior cruciate ligament (ACL) injury, a sports-related injury, affects young to middle-aged patients and in the United States occurs in approximately 200,000 patients per year.¹³ A lack of appropriate treatment for ACL ruptures puts patients at risk for future osteoarthritis,³⁸ and treatment options range from nonsurgical to surgical, with the latter often preferred by high-performance athletes.²³ To date, ligaments have been reconstructed using artificial ligaments, autologous hamstring tendon or patellar tendon grafts, or allografts, such as the iliotibial band.⁴ However, given the poor long-term results seen after reruptures of reconstituted ligaments, reconstruction procedures using autologous tendons, such as hamstring tendon grafts, bone–patellar tendon–bone grafts, or quadriceps tendon autografts, are now performed globally.^11,31 Nonetheless, even this treatment requires 6 to 10 months of recovery before patients can return to sports activities,^31,36 and postoperatively, tendon graft strength temporarily decreases.²⁶ Top athletes want to return to sports activities more rapidly; thus, the development of treatment methods to speed up the recovery time from surgery is an area of great interest.^14,31

Recently, the number of studies concerning mesenchymal stem cells (MSCs) in the field of orthopaedics has increased.^3,10 In particular, bone marrow–derived MSCs (BM-MSCs) have been extensively applied to treat disorders of tendons, ligaments, and bone.^5,7 However, collecting BM-MSCs from bone marrow is highly invasive, and even if stem cells are collected, their number is often small, and considerable time is required to expand them before transplantation.¹² Meanwhile, cells known as adipose-derived stem cells (ADSCs) are present in adipose tissue,⁵³ and compared with BM-MSCs, ADSC collection is less surgically invasive. Currently, it is possible to collect large numbers of ADSCs for application to orthopaedic problems, such as knee osteoarthritis or meniscal tears.^2,25,44

Okano et al³⁵ developed the cell sheet dish as a tissue-engineering technique. Generally, while cells on a cell culture dish cannot be detached without treatment with enzymes such as trypsin, this specialized dish allows the detachment of contiguous cell sheets with intact cell-cell connections and the extracellular matrix simply by temperature reduction without any scaffold. The technique has been applied to esophageal and cardiovascular diseases in Japan, and clinical trials are showing promising results.^19,34,41 Although comparable approaches have been applied in the field of orthopaedics, such as in cartilage regeneration,^39,40 bone regeneration,^6,49 and tendon repair,²⁰ studies of its use for the ACL are limited.^5,32 Hence, we hypothesized that applying ADSC sheets around transplant grafts during ACL reconstruction (ACLR) would improve both biomechanical strength and tendon-bone healing. Thus, the purpose of this study was to report the effects of ADSC sheets on grafts for ACLR.

Methods

Study Design

ACLR was performed on the right knees of 84 twenty-week-old female Japanese White rabbits with either the addition or omission of cell sheets. Animals were followed for 2, 4, 8, 16, or 24 weeks, and overall, 68 rabbits were analyzed; exclusions included 6 rabbits because of complications from a patellar dislocation, 5 because of poor bone tunnel positioning, 3 for intraoperative graft creation problems, 1 for a biomechanical examination problem, and 1 because of death. Rabbits were randomly divided for biomechanical testing, micro–computed tomography (µCT), or histological staining. Among the 68 rabbits, biomechanical testing and µCT were performed in 44 (including six 24-week-old rabbits for which scanning transmission electron microscopy [TEM] was performed). 10, 10, 8, 8, or 8 rabbits were analyzed at 2, 4, 8, 16, or 24 weeks after surgery, respectively, by both biomechanical testing and μCT. Histopathological analyses were conducted in 24 rabbits. Each 6 rabbit was analyzed histopathologically at 4, 8, 16, or 24 weeks after surgery.

Rabbits

Rabbits were purchased from CLEA Japan and placed under specific pathogen-free conditions in animal facilities certified by our Institutional Animal Care and Use Committee. Animals were maintained in an environment in accordance with our institutional guidelines on animal experimentation. All animal experimental protocols were approved by the aforementioned committee and performed in accordance with its guidelines.

ADSC Sheets

ADSCs (StemPro Human Adipose-Derived Stem Cells [Lot #1001002]; Invitrogen) used in this study were expanded from P0 and cryopreserved at P3 in STEM-CELLBANKER (Nippon Zenyaku Kogyo). Cells were then recultured, and at P4, when cells had expanded uniformly, 6-cm UpCell plates (CellSeed) were seeded with 4 × 10⁵ cells and cultured for 4 days until overconfluence. Dishes were then placed at room temperature for cooling. Then, cells were detached from the dishes as sheets without a scaffold, and all cell sheets were uniformly constructed. The cell number per sheet was determined in separate dishes after collecting the cultured cells after trypsinization, and the number was approximately 1 × 10⁶ cells per sheet. During culture, L-Ascorbic Acid Phosphate Magnesium Salt n-Hydrate (FUJIFILM Wako Pure Chemical) was added at 0.1 mg/mL to the medium (MesenPRO RS Medium; Thermo Fisher Scientific) without fetal bovine serum. Cell sheets were washed with saline before use.

Surgery

All surgical procedures were performed by an author (T. Matsumoto), who was assisted by a co-author (K.S.). Ketamine hydrochloride (35 mg/kg; Daiichi Sankyo) and xylazine hydrochloride (5 mg/kg; Bayer) were subcutaneously administered at the posterior region of the neck of the 20-week-old rabbits. The rectal administration of buprenorphine (0.2 mg; Otsuka Pharmaceutical) was performed along with the intramuscular administration of the antibiotic enrofloxacin (7 mg/kg; Bayer) into the upper right limb. The surgical area was shaved, disinfected, and draped, followed by the administration of local anesthesia with lidocaine hydrochloride (Maruishi Pharmaceutical) just before incision. A 5-cm medial incision was made, and 1 cm of the inner fascial membrane was cut, after which the semitendinosus tendon (ST) was identified and a 3- to 4-cm graft was obtained (Figure 1, A and B). After separating the muscles, baseball sutures were applied at both ends of the graft using 4-0 Vicryl Plus sutures (Johnson & Johnson). A cell sheet of approximately 1 cm was then wrapped around the center of the graft in the sheet group (Figure 1C). The midvastus approach was used to expose the inner joint with the patella laterally dislocated; the original ACL was then excised. A 2.0-mm drill was used to create bone tunnels on the tibia and femur at the original footprint of the ACL (Figure 1D). Because of concerns about damage to the sheet during graft passage through the bone tunnel, the graft was passed inside-out from the inner part of the joint toward the outside (Figure 1E). The graft was then sutured to the periosteum of the femur and tibia at neutral tension with the knee in 90° of flexion using 4-0 Vicryl Plus sutures (Figure 1F). In the sheet group, another sheet was wrapped around the graft portion within the joint just before closure to ensure that the entire graft was wrapped by the sheet. Cell sheets have been shown to exhibit adhesiveness to tissue such as the esophagus and even beating hearts without a scaffold or adhesive^34,41; thus, in our study, cell sheets were secured on the ACL graft without a scaffold or adhesive. Joint closure was performed after confirming that there were no restrictions in range of motion, and surgery was completed by closing the skin using Dermabond Advanced (Johnson & Johnson).

Biomechanical Examination

Soft tissues around the knee, including the meniscus, posterior cruciate ligament, medial collateral ligament, and lateral collateral ligament, were immediately excised after animal sacrifice, and only the bones and transplanted graft were cryopreserved at −80°. These were thawed at ambient temperature before undergoing an examination with an Autograph AG-IS tensile testing machine (Shimadzu). The ultimate failure load (UFL) was measured by setting a K-wire to the machine and to the tibia and femur in a neutral position at a speed of 10 mm/min. The UFL and stiffness were measured based on experiment-derived curve results.

Histological Evaluation

After sacrifice, samples for pathological analysis were fixed in 10% neutral formalin for 3 days and decalcified with ethylenediaminetetraacetic acid for 5 weeks. Histological slides were prepared by assembling slices of the joint along the femoral tunnel axis, and slides were stained using Masson trichrome. Bright-field TEM was performed to examine histological changes in the healing process of the tendon-bone interface and graft midsubstance. A previously reported scoring system was used (Table 1).^26,42

Table 1

Histological Scoring System for Tendon-Bone Healing

	Score
1. Cellular morphology of interface tissue
Fibrocartilage with mature cartilage cells comprising ≥50%	4
Fibrocartilage with mature cartilage cells comprising <50%	3
Fibrous tissue with Sharpey-like fibers comprising ≥50%	2
Fibrous tissue with Sharpey-like fibers comprising <50%	1
Only fibrovascular tissue	0
2. Extent of fibrocartilage tissue
Mostly surrounded (≥75%)	4
Moderately surrounded (≥50% to <75%)	3
Partially surrounded (≥25% to <50%)	2
Slightly surrounded (<25%)	1
Not visible	0
3. Interface tissue transition from bone to tendon
Mostly indistinct (≥75%)	4
Moderately indistinct (≥50% to <75%)	3
Partially indistinct (≥25% to <50%)	2
Continuous but slightly indistinct (<25%)	1
Discontinuous	0

µCT Analysis

Images of the femur were obtained directly before biomechanical testing. µCT was performed using R_mCT2 (Rigaku) with the following parameters: field of view, 30 mm; voltage, 90 kV; current, 160 mA; and scan time, 3 minutes (60 µm/slice; 433 pixels/inch). TRI/3D-BON (Ratoc Systems) and ImageJ (Version 1.52; National Institutes of Health) software were used to measure bone volume/tissue volume (BV/TV) and bone tunnel cross-sectional area (bone tunnel area). The bone tunnel area was divided into 4 equal parts, with the portion closest to the articular surface defined as the joint opening, and the cross-sectional area of this part was measured (Figure 2A). The BV/TV was measured with a volume of 3.5 mm³ at the center of the femoral tunnel (Figure 2B).

Scanning TEM

After biomechanical analysis, at 24 weeks after surgery, the undamaged graft and native ST from the contralateral limb were fixed in 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer (PB; pH, 7.4) overnight at 4°C. Samples were then washed 3 times with 0.1 M PB for 30 minutes each and fixed with 2% osmium tetroxide in 0.1 M PB overnight at 4°C. Samples were dehydrated in graded ethanol solution and then continuously dehydrated in 100% ethanol at room temperature overnight. The samples were infiltrated with propylene oxide (PO) twice for 1 hour each and placed in a 70:30 mixture of PO and resin (Quetol-812; Nisshin EM) for 2 hours. Then, the tube cap was kept open, and PO was volatilized overnight. Samples were transferred to fresh 100% resin and polymerized at 60°C for 48 hours. Blocks were ultrathin-sectioned at 70 nm with a diamond knife using an ultramicrotome (Ultracut UCT; Leica), and sections were placed on copper grids. The grids were observed using TEM (JEM-1400Plus; JEOL) at an acceleration voltage of 100 kV. Digital images (3296 × 2472 pixels) were obtained using a charge-coupled device camera (EM-14830RUBY2; JEOL). Moreover, 10 TEM images were randomly obtained by blinding each sample and analyzed using an image analysis system (Version 1.52; ImageJ). For analysis, particle homogenization and contrast were first enhanced and then binarized while visually confirming the original drawing, and particles were smoothed, underwent watershed separation, and measured. To evaluate fibril diameters, the minimum fibril diameter was measured to prevent errors due to obliquity in sectioning. Fibril structures with a diameter <10 nm were excluded to avoid measuring microfibrils or noncollagen fibrillar components. The number of collagen fibers, fibril diameters, and occupation ratio of collagen fibers per field of view were calculated.

Statistical Analysis

All statistical analyses were performed using Prism 8 software (GraphPad Software). Bar and line graph data were expressed as the mean ± SD. Boxplot values were presented as the minimum, maximum, median, and first and third quartiles. The Welch t test was used to compare the control and sheet groups at each time point. Pearson correlation analysis was performed for associations between biomechanical findings and the BV/TV, and analysis of variance was used for TEM analysis. P values <.05 indicated statistical significance.

Results

Biomechanical Examination

Overall, 20-week-old rabbits underwent unilateral ACLR with an ST autograft, with or without cell sheets. Over a period of 2 to 24 weeks, the UFL was analyzed at various time points (Figure 3A). The median UFL for the control and sheet groups, respectively, was as follows: 17.2 ± 5.5 versus 37.3 ± 10.3 (P = .01) at 2 weeks, 28.6 ± 1.9 versus 47.4 ± 10.4 (P = .003) at 4 weeks, 53.0 ± 14.3 versus 48.1 ± 9.3 (P = .59) at 8 weeks, 66.2 ± 9.3 versus 95.2 ± 43.1 (P = .24) at 16 weeks, and 66.7 ± 27.3 versus 85.3 ± 29.5 (P = .39) at 24 weeks. Except at 8 weeks, the UFL was higher in the sheet group relative to the control group on biomechanical testing, a difference that was statistically significant in the early stages up to 4 weeks (Figure 3B). Median stiffness values for the control and sheet groups, respectively, were as follows: 7.7 ± 0.7 versus 13.1 ± 2.5 (P = .01) at 2 weeks, 14.6 ± 4.5 versus 18.0 ± 5.3 (P = .31) at 4 weeks, 17.5 ± 5.8 versus 16.2 ± 4.9 (P = .74) at 8 weeks, 24.8 ± 7.0 versus 28.7 ± 6.0 (P = .43) at 16 weeks, and 24.0 ± 4.2 versus 26.5 ± 6.0 (P = .52) at 24 weeks. Stiffness was also significantly higher in the sheet group relative to the control group at 2 weeks postoperatively (Figure 3C). The number of animals with midsubstance failure was higher in the sheet group than the control group at every interval (Table 2).

Table 2

Failure Pattern on Biomechanical Testing

	Control Group, n		Sheet Group, n		Midsubstance Tear Ratio, %
	Midsubstance Failure	Pullout Failure	Midsubstance Failure	Pullout Failure	Control Group	Sheet Group
2 wk		5		5	0	0
4 wk		5	1	4	0	20
8 wk	1	3	2	2	25	50
16 wk	2	2	3	1	50	75
24 wk	3	1	4		75	100

Histological Evaluation

At the tendon-bone interface, Sharpey-like fibers and the emergence of hypertrophic chondrocytes were more pronounced in the sheet group than the control group at 4 weeks after surgery (Figure 4, A and E). At 8 weeks, osteoid formation was more frequently observed in the sheet group, and the tendon-bone interface was less disorganized in the sheet group relative to the control group (Figure 4, B and F). At 16 and 24 weeks, histological findings were equivalent between the sheet and control groups (Figure 4, C, D, G, and H). As a result, the histological score determined by cellular morphology of interface tissue, extent of fibrocartilage tissue, and interface tissue transition from bone to tendon was significantly higher in the sheet group than the control group at 4 and 8 weeks after surgery, although scores between groups were comparable at 16 and 24 weeks after surgery. Histological scores for the control and sheet groups, respectively, were as follows: 3.0 ± 0.9 versus 4.2 ± 0.8 (P = .04) at 4 weeks, 5.7 ± 0.5 versus 6.5 ± 0.5 (P = .02) at 8 weeks, 8.2 ± 0.4 versus 8.5 ± 0.5 (P = .26) at 16 weeks, and 8.7 ± 0.5 versus 9.0 ± 1.1 (P = .52) at 24 weeks (Figure 5).

µCT Analysis

Bone characteristics in rabbit femurs after ACLR with or without cell sheets were evaluated by µCT at indicated time points after surgery (Figure 6). The femoral bone tunnel, created for ACLR, was divided into 4 equal parts with the nearest cross section to the joint defined as the joint opening, and the cross-sectional area of this site was measured in the control and sheet groups. The obtained values for the control and sheet groups, respectively, were as follows: 3.9 ± 1.1 versus 3.5 ± 1.1 (P = .61) at 2 weeks, 7.3 ± 1.0 versus 5.1 ± 0.9 (P = .01) at 4 weeks, 6.6 ± 2.1 versus 4.7 ± 0.8 (P = .25) at 8 weeks, 4.3 ± 0.7 versus 4.6 ± 1.0 (P = .64) at 16 weeks, and 4.3 ± 1.1 versus 4.1 ± 1.5 (P = .53) at 24 weeks (Figure 6A). The cross-sectional area of the joint opening was significantly smaller in the sheet group than the control group at 4 weeks postoperatively, but there was no significant difference at the other time points analyzed. The BV/TV (%) at the tendon-bone interface with either calcification of >400 mg/mL or ≥800 mg/mL increased over time in both the sheet and control groups but was significantly higher in the sheet group at 24 weeks after ACLR (Figure 6, B and C). The results of calcification >400 mg/mL for the control and sheet groups, respectively, were as follows (Figure 6B): 21.9 ± 6.0 versus 20.1 ± 5.9 (P = .65) at 2 weeks, 21.5 ± 3.7 versus 22.8 ± 2.0 (P = .53) at 4 weeks, 29.5 ± 1.3 versus 24.4 ± 3.5 (P = .06) at 8 weeks, 33.5 ± 2.6 versus 38.4 ± 4.6 (P = .11) at 16 weeks, and 32.4 ± 4.4 versus 43.4 ± 8.2 (P = .05) at 24 weeks. When calcification was analyzed at ≥800 mg/mL, the results for the control and sheet groups, respectively, were as follows: 0.4 ± 0.2 versus 0.3 ± 0.2 (P = .56) at 2 weeks, 0.7 ± 0.7 versus 1.3 ± 0.8 (P = .29) at 4 weeks, 1.2 ± 0.8 versus 2.6 ± 2.2 (P = .35) at 8 weeks, 3.3 ± 1.8 versus 6.8 ± 4.6 (P = .21) at 16 weeks, and 4.0 ± 3.4 versus 10.0 ± 1.8 (P = .02) at 24 weeks (Figure 6C). Excluding cases of midsubstance ruptures, correlation analysis between the BV/TV (>400 mg/mL) and UFL of samples experiencing pullout revealed a positive correlation (R = 0.72) between the groups (P = .0002) (Figure 7).

TEM Analysis

Finally, TEM analysis was performed at 24 weeks postoperatively (Figure 8A). The total number of fibers per field of view in the native ST from the contralateral rabbit limb at that time point was 333 ± 125, while in the control and sheet groups, the numbers were 527 ± 235 and 473 ± 175, respectively, with no significant difference (Figure 8B). However, each fibril diameter (minimum Feret diameter) was 90 ± 16nm in the native ST, and this value was significantly greater in the sheet group (58.7 ± 56.8nm) compared with the control group (50.5 ± 51.5nm) (P < .001) (Figure 8C). For histograms of each fibril diameter, a composition closer to the native ST was obtained in the sheet group (Figure 8D). The occupation ratio of collagen fibers per field of view of the native ST was 64.0 ± 1.3, while this ratio was significantly greater in the sheet group (54.5 ± 11.0) compared with the control group (47.4 ± 14.0) (Figure 8E).

Discussion

It is currently difficult for athletes to return to sports activities soon after ACLR. Our study demonstrates that the application of an ADSC sheet during surgery improved early biomechanical strength and promoted healing of the tendon-bone interface and graft midsubstance, suggesting that ADSC sheet transplantation may enable an earlier return to sports after ACLR.

Recent studies have reported on the application of various types of stem cells,¹⁰ including MSCs,⁴³ tendon-derived stem cells (tenocytes),³⁰ and ADSCs, in ACLR.²¹ In these studies, such cells are either directly injected into the joint,³² injected into the joint after mixing with fibrin glue,¹⁶ or formed into a sheet and transplanted.¹⁸ Other investigators have applied cell sheets to tissue, such as the articular cartilage, esophagus, and heart, and shown that they promote tissue repair and/or functional improvement.^34,39,41 ADSCs reportedly differentiate into osteochondrocytes^2,25,28,52 or tendon cells,^22,27 depending on the environment, and occasionally result in ectopic ossification as an adverse outcome.⁸ Kosaka et al²¹ and Zhang et al⁵⁰ injected ADSCs into the joint space, although the localization of transplanted cells in joints was unclear. In our study, an ADSC sheet was wrapped around the graft, allowing it to potentially release trophic factors directly onto the graft. Nonetheless, at present, it is still not known when a graft is “strong enough” to resume a particular sports activity and what loads the graft is capable of withstanding with specific sports activities. Although there is a decline in graft strength, how the actual material properties of the graft change over time is also unclear. Moreover, the timetable for returning to sports activities depends on factors other than graft strength, including the restoration of muscle strength, endurance, balance, coordination, and proprioception.

After ACLR using hamstring tendon grafts, bone tunnel widening has been reported, leading to graft loosening and the necessity for revision surgery.^9,46 At an early stage in ACLR, the bone tunnel reportedly exhibits transient widening,⁴⁸ and 1 study reported that bone tunnel widening may be prevented by MSC transplantation.¹⁵ In the current study, bone tunnel widening was significantly prevented in the sheet group compared with the control group at 4 weeks postoperatively. This effect likely resulted in a significantly higher UFL at an early stage, as shown in the biomechanical examination findings, except at 8 weeks. The prevention of bone tunnel widening further caused a high number of midsubstance graft ruptures in the sheet group likely because of enhanced enthesis formation and tight bone-graft interaction, although the collagen fiber diameter was significantly thickened by ADSC sheets. Thus, we conclude that improved graft-to-bone healing shifted the failure site to the graft midsubstance.

Sharpey-like fibers reportedly function to connect bone with either tendons, ligaments, or grafts and are required to strengthen those connections.²¹ Here, we demonstrated that the formation of Sharpey-like fibers and the emergence of hypertrophic chondrocytes were more pronounced in the sheet versus control group. It is possible that the trophic activity of ADSCs promoted healing of the tendon-bone interface or that ADSCs themselves functioned as living cells in the graft midsubstance. Although results from biomechanical testing showed an increase in the frequency of midsubstance ruptures over time in both groups, the use of ADSCs promoted bone tissue formation with a higher BV/TV in the sheet group. This increase in the ossification of the tendon-bone interface over time could strengthen the graft and/or bone adhesion, reducing the incidence of reconstructed graft pullout. In a report on ADSC gel transplantation during surgery for chronic rotator cuff tears, the authors reported an improvement in bone mineral density at the tendon-bone interface,¹⁷ suggesting that ADSCs stimulate bone-forming activities.

Takeuchi et al⁴⁵ showed that the average collagen fiber thickness of a transplant graft shrinks over time and that the composition ratio of collagen fibers decreases. Furthermore, it is reported that after surgery, newly synthesized fibers are thin because of their type III collagen appearance.³³ Our TEM analysis of the midsubstance demonstrated that the use of ADSC sheets resulted in a larger mean diameter of each fibril compared with that seen in the control group. The presence of type I collagen reportedly enlarges the collagen fiber diameter⁴⁵; thus, ADSC sheets may promote the synthesis of type I collagen. Moreover, the composition ratio of collagen fibers per cross-sectional area increased in the sheet group, and this outcome may increase the UFL during midsubstance ruptures. As previous studies reported that a rerupture after ACLR occurs in the midsubstance rather than at the tendon-bone interface,^29,37 strengthening the midsubstance by the incorporation of ADSCs may decrease the rate of reruptures. These outcomes may be caused by the expression of trophic factors that promote tissue repair³⁹ and enhance collagen fiber maturation, although at present, mechanisms underlying observed increases in the collagen fiber diameter remain unclear. Further research is required to define the mechanisms underlying the improvement of the biomechanical strength of ACLs reconstructed with ADSC sheets.

Biological structures and surgical techniques used to repair the knees of rabbits and humans differ and may account for some of our study’s limitations. Specifically, surgical techniques differ because of the body size of humans versus rabbits. In rabbits, the graft was sutured to the periosteum for fixation, which may have caused pullout failure of the grafts. We note, however, that failure rates reported here were comparable with those described by others using the same technique in rabbits.²⁶ We observed surgical failure such as patellar dislocations and poor bone tunnel positioning again likely because of the small size of the animals, and these rabbits were excluded from the study. Moreover, biological responses to xenotransplantation of human ADSC sheets into rabbits also remain unclear. MSCs, including ADSCs, reportedly have immune-suppressive effects,^1,24 and allografts or xenografts have been transplanted without immune-suppressive agents.^47,51 Overall, however, we believe that our results illustrate that ADSC sheet application in ACLR improves biomechanical strength, promotes early maturation of the tissue structure at the tendon-bone interface, and maintains thicker collagen fibers in the graft midsubstance. We note, however, that the collagen fiber diameter was measured at 1 time point. Neither creating a cell sheet from adipose tissue nor wrapping the sheet during ACLR is technically difficult, and both are considered clinically appropriate techniques, as cell sheets have already been applied to treat human diseases such as esophageal and cardiovascular diseases.^34,41 The application of an ADSC sheet to a graft could be of significant value to high-performance athletes after an ACL rupture.

Clinical Relevance

Our data demonstrate that the biomechanical strength of the reconstructed ACL improved at an early stage after surgery with the application of an ADSC sheet. The clinical application of ADSC sheets may allow athletes to return to sports at earlier postoperative time points. Furthermore, rerupture rates may be decreased by wrapping ADSC sheets around a graft.

Conclusion

ADSC sheet application contributed to early improvement in mechanical strength, promoted early maturation of the tendon-bone interface in ACLR, and prevented bone tunnel widening after surgery.

Authors

Tatsuaki Matsumoto, MD (Department of Orthopedic Surgery, School of Medicine, Keio University, Tokyo, Japan); Yuiko Sato, MB (Department of Orthopedic Surgery, School of Medicine, Keio University, Tokyo, Japan; Department of Advanced Therapy for Musculoskeletal Disorders II, School of Medicine, Keio University, Tokyo, Japan; Department of Musculoskeletal Reconstruction and Regeneration Surgery, School of Medicine, Keio University, Tokyo, Japan); Tami Kobayashi, BS (Department of Orthopedic Surgery, School of Medicine, Keio University, Tokyo, Japan; Department of Advanced Therapy for Musculoskeletal Disorders II, School of Medicine, Keio University, Tokyo, Japan; Department of Musculoskeletal Reconstruction and Regeneration Surgery, School of Medicine, Keio University, Tokyo, Japan); Kunika Suzuki, DVM (Department of Orthopedic Surgery, School of Medicine, Keio University, Tokyo, Japan; Regenerative Medicine iPS Gateway Center, Tokyo, Japan); Atsushi Kimura, MD (Department of Orthopedic Surgery, School of Medicine, Keio University, Tokyo, Japan); Tomoya Soma, DDS, PhD (Division of Oral and Maxillofacial Surgery, Department of Dentistry and Oral Surgery, School of Medicine, Keio University, Tokyo, Japan); Eri Ito, MD, PhD (Institute for Integrated Sports Medicine, School of Medicine, Keio University, Tokyo, Japan); Toshiyuki Kikuchi, BS (Department of Clinical Research, National Hospital Organization Murayama Medical Center, Tokyo, Japan); Shu Kobayashi, MD, PhD (Department of Orthopedic Surgery, School of Medicine, Keio University, Tokyo, Japan); Kengo Harato, MD, PhD (Department of Orthopedic Surgery, School of Medicine, Keio University, Tokyo, Japan); Yasuo Niki, MD, PhD (Department of Orthopedic Surgery, School of Medicine, Keio University, Tokyo, Japan); Morio Matsumoto, MD, PhD (Department of Orthopedic Surgery, School of Medicine, Keio University, Tokyo, Japan); Masaya Nakamura, MD, PhD (Department of Orthopedic Surgery, School of Medicine, Keio University, Tokyo, Japan); and Takeshi Miyamoto, MD, PhD (Department of Orthopedic Surgery, School of Medicine, Keio University, Tokyo, Japan; Department of Advanced Therapy for Musculoskeletal Disorders II, School of Medicine, Keio University, Tokyo, Japan; Department of Musculoskeletal Reconstruction and Regeneration Surgery, School of Medicine, Keio University, Tokyo, Japan; Department of Orthopedic Surgery, Kumamoto University, Kumamoto, Japan).

Footnotes

Acknowledgements

The authors thank Kunika Suzuki for her assistance during surgery.

Submitted January 11, 2021; accepted May 24, 2021.

One or more of the authors has declared the following potential conflict of interest or source of funding: K.S. is an employee of Regenerative Medicine iPS Gateway Center. This study was supported by Regenerative Medicine iPS Gateway Center. AOSSM checks author disclosures against the Open Payments Database (OPD). AOSSM has not conducted an independent investigation on the OPD and disclaims any liability or responsibility relating thereto.

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