Engineered Decellularized Tendon Matrix Putty Preserves Native Tendon Bioactivity to Promote Cell Proliferation and Enthesis Repair

Rotator cuf tears are a common soft tissue injury that can signifcantly decrease function of the shoulder and cause severe pain. Despite progress in surgical technique, rotator cuf repairs (RCRs) do not always heal efciently. Many failures occur at the bone-tendon interface as a result of poor healing capacity of the tendon and failure to regenerate the native histological anatomy of the enthesis. While allografts are commercially available, clinical use is limited as they do not stimulate tissue regeneration and are associated with a structural failure of up to 40% in re-tear cases. Novel tissue engineering strategies are being developed with promise, but most involve addition of cells and/or growth factors which extends the timeline for clinical translation. Tus, there exists a signifcant unmet clinical need for easily translatable surgical augmentation approaches that can improve healing in RCR. Here we describe the development of a decellularized tendon matrix (DTM) putty that preserves native tendon bioactivity using a novel processing technique. In vitro , DTM promoted proliferation of tenocytes and adipose-derived stem cells with an increase in expression-specifc transcription factors seen during enthesis development, Scleraxis and Sox9 . When placed in a rabbit model of a chronic rotator cuf tear, DTM improved histological tissue repair by promoting calcifcation at the bone-tendon interface more similar to the normal fbrocartilaginous enthesis. Taken together, these data indicate that the engineered DTM putty retains a pro-regenerative bioactivity that presents a promising translational strategy for improving healing at the enthesis.


Introduction
Tendon and ligament injuries afect approximately 17 million Americans each year and are the second leading cause of musculoskeletal injuries [1].In 2016, there were over 460,000 rotator cuf surgeries performed in the United States and it was the second most common orthopaedic soft tissue repair procedure performed [2].Despite signifcant clinical progress to improve surgical technique and rehabilitation protocols, structural outcomes of rotator cuf repair (RCR) remain statistically poor.Re-tear rates as high as 40% have been noted following standard rotator cuf repairs with failures increasing to 73-94% in massive tears [3][4][5][6][7][8][9][10]. In addition to the higher re-tear rates with increased tear size, there is also an increased incidence of repair failures with increasing patient age [11].
Augmenting repair at the tendon-bone junction may improve RCR as this is where the vast majority of postoperative failures occur [5,12].Insertion of the tendon into the bone at the rotator cuf occurs through a fbrocartilaginous enthesis where there is a gradual histological transition from tendon to fbrocartilage to calcifed cartilage to bone [13].Adult entheses do not regenerate following RCR, rather repair typically produces a mechanically weak fbrovascular scar lacking the zone of calcifed cartilage [14][15][16].A number of critical factors likely contribute to the poor regenerative capacity of the tendon, including a paucity of appropriate tendon progenitor cells, the low vascularity of the fbrocartilaginous enthesis, and limited bioavailability of growth factors that can promote regeneration.Given the poor clinical results of RCR, there is currently a major challenge in the feld of orthopaedic medicine to stimulate biological healing and reconstruct the enthesis structure to regenerate a stronger tendon.
Interestingly, while adult tendon tissues heal poorly, neonatal tendons can successfully regenerate following injury in a process termed "scarless healing" [17,18].Lineage tracing identifed Scleraxis-(Scx-) positive tendon progenitor cells as the key cellular population enabling neonatal tendon development [19,20].Recent work has further observed that co-expression of Scx and transcription factor Sox9 is critical to enthesis development and repair [21,22].In adult RCR, these Scx + /Sox9 + cells are not recruited to the injury and there is minimal cellular proliferation after injury [18,21].In the adult tissue, these Scx-lineage tenocytes are not recruited to the injury, and there is very minimal cellular proliferation 3 days after injury [18].Regulation of tenocyte recruitment and direction of collagen fbrils at the enthesis have been reported to be directly regulated by Transforming Growth Factor-β (TGFβ) signaling [23,24].Regulation of tenocyte recruitment and the subsequent functional tendon regeneration appear to be directly regulated by Transforming Growth Factor-β (TGFβ) signaling in the neonatal tissue [23].
In this preliminary investigation, we develop and characterize a decellularized tendon matrix (DTM) putty utilizing a novel enzymatic processing technique which promotes tenocyte maturation, preserves TGFβ bioactivity, and promotes cell proliferation.Decellularized matrices have been utilized extensively in bone regeneration strategies to provide a biomimetic and bioactive substrate to support structural and biological healing.Tendon and dermal allografts are commercially available to support RCR, but their use has been limited due to the combination of unclear impact on clinical outcomes and limited evidence of bony ingrowth from the tuberosity into the allograft [25,26].Engineered decellularized matrices provide an opportunity to improve upon allograft technology by preserving the bioactivity of the tissue and generating a clinically more useful form factor for surgical application.We hypothesized that our novel method for creating a decellularized tendon matrix (DTM) putty would maintain bioactivity of the native tissue to enhance tendon-to-bone healing model.

Tendon Allograft and Tendon
Processing.Achilles and patella tendon allografts were donated from Musculoskeletal Transplant Foundation (MTF, Edison, NJ) using the MTF Biologics Non-Transplantable Tissue Program.A total of 10 donors (5 males and 5 females with ages ranging from 18 to 61) were used in this study.Tendons were delivered on dry ice, thawed, and dissected into proximal, mid, and distal thirds for regional characterization (Figure 1(a)).Te tendon was minced into smaller pieces and then decellularized using a tissue processing method involving DNase I at 50 U per 1 mL of 1X PBS in solution for 1 hour at 56 °C with moderate shaking.Decellularized tendons were washed in excess PBS, frozen, and lyophilized until dry.DTM processing was compared to standard decellularization detergents sodium dodecyl sulfate (SDS, 1%) and ethylenediaminetetraacetic acid (EDTA, 0.1%) [27].Te untreated tendon was treated with PBS.DNA was isolated using DNeasy Blood and Tissue Kit per manufacturer's protocol (n = 2 donors/group).Te DNA isolate was measured with Tecan's Nano Quant Plate analyzed using the 260/280 ratio read by Tecan's Infnite 200 Pro Plate reader.To develop an injectable tendon putty, 0.275 g of the lyophilized and decellularized tendon was placed in 1 mL of collagenase solution (collagenase type I at 2 mg/mL and collagenase type III at 1 mg/mL in 1X PBS) for enzymatic digestion.Te tendons were enzymatically digested in this solution for 12 hours at 37 °C with shaking and were then frozen and lyophilized.To remove collagenase solution from DTM, the tendons were reconstituted and placed in 100K molecular weight cutof (MWCO) protein concentrators, spun at 8, 000 × g for 5 minutes.Inactivation of the enzymatic digestion was verifed using a Collagenase Activity Colorimetric Assay Kit, performed according to manufacturer's protocol.Te reaction plate was quantifed using Tecan's Infnite 200 Pro Plate reader at an absorbance of 345 nm.Collagenase activity was graphed as U/mg of the tendon (n = 3-6).

Protein Isolation, Quantifcation, and TGFβ Protein
Analysis.To obtain protein isolates, T-PER was added to the donor tendon samples with Protease Inhibitor Cocktail.Te samples were homogenized using a tissue homogenizer and placed at 4 °C for 2 hours to allow for protein extraction.Te samples were fltered using 70 μm cell strainers spinning at 2, 000 × g for 10 minutes.Total protein was quantifed using a disulfde reducing agent compatible microplate bicinchoninic acid assay (Micro-BCA) according to manufacturer's protocol.Protein concentrations of the experimental values were calculated using a linear model in micrograms of protein per milliliter of diluted sample.Protein isolates were used to quantify TGFβ using TGFβ Magnetic Bead 3 Plex Kit.A Luminex 200 ™ Instrument System was used to detect the analytes according to the Bead Panel's manufacturer protocol.30 mg of total protein was placed within each of the wells for TGFβ analysis, and the fnal output is expressed in picograms of TGFβ per mL of sample.

2
Journal of Tissue Engineering and Regenerative Medicine Standard culture conditions were used as the control for both tenocytes and ADSCs (collagen-coated and tissueculture treated, respectively).Collagen coating was done using collagen I at a concentration of 0.  skin incision was made, the supraspinatus tendon was detached from the greater tuberosity, and a Penrose drain was inserted into the free end of the tendon to prevent its spontaneous reattachment.Te fascia was closed using a 3-0 absorbable Vicryl sutures, and the wound was closed with 4-0 sutures.Te rabbits were allowed regular activity for 6 weeks to develop a chronic tendon tear model.After 6 weeks, the rabbits underwent tendon repair surgery.Using the same anesthesia and sterilization techniques as previously stated, the Penrose was identifed and removed, and the supraspinatus tendon was fxed to the footprint at the greater tuberosity through 2 transosseous tunnels.Te

Allograft Sourcing for Decellularized Tendon Matrix (DTM).
Achilles and patellar tendons are the most readily available, abundant, and accessible allograft tendon tissues.It was unclear whether the protein content is diferent between these two tendon sources or whether sexual dimorphism is signifcant.No signifcant diference was observed in total protein content between Achilles and patellar tendons (Figure 3(a)) or across sex (Supplemental Figure 1).Since the vascularity of tendons decreases in the central region, in what is often described as the "watershed zone," we aimed to further understand if protein content varied by location within the tendon.Achilles and patellar allografts were grossly dissected into proximal, midcenter, and distal segments based on dividing the total tendon length into thirds (Figure 3(b)).No signifcant diference in protein content was noted between proximal, midcenter, and distal divisions within tendons (Figures 3(c) and 3(d)).On this basis, decellularization of the tendon was performed on the composite of the whole Achilles and patellar tendon moving forward.

Decellularization Step Efectively Removes Cellular
Content.To develop a method for efective decellularization without loss of matrix proteins and bioactivity, we compared an enzymatic decellularization protocol for DTM to previously published detergent-based techniques that used 1% SDS or 0.1% EDTA for 24 hours [27,32].All decellularization techniques efectively removed DNA relative to PBS (Figure 1(a)), with Tukey's HSD multiple comparison post hoc testing fnding no signifcant diference between DNA content following decellularization using the DTM process compared to SDS or EDTA (Figure 1(a)).Removal of DNA was confrmed histologically by sectioning and staining with DAPI (Figures 1(b) and 1(c)).

Tendon Processing Maintains Protein Content and Creates a Tendon Putty.
To enhance the surgical usability of DTM, we next enzymatically digested the decellularized tendon allograft using collagenase to break up the dense matrix and create a moldable form factor. Te optimized digestion protocol for DTM generated a viscoelastic putty that can be stretched and reformed but maintained structural integrity (Figures 4(a) and 4(b)).Rheological properties of the DTM product were measured across various reconstitution concentrations (1 gram of tendon diluted in 1, 3, 5, or 7 mL PBS) to determine sample rigidity and structure strength.Te oscillation stress sweeps reveal that samples at all dilutions display elastic dominant behavior across the full range of frequencies applied with overlapping mechanical properties at 3 and 5 mL dilutions (Figure 4(c)).Te complex modulus plateaus (sample rigidity) and yield stress (sample strength) values decreased with increased dilution (Table 2).Putty-like gross structural properties were maintained in dilutions of 1 gram in 1-5 mL PBS; however, 7 mL exceeded the Journal of Tissue Engineering and Regenerative Medicine saturation limit of the tissue putty.Since these mechanical properties were achieved by enzymatically digesting the decellularized tendon allograft, we next aimed to ensure that enzymes were sufciently removed to stop the digestion reaction.We found fltration of the digested allograft was sufcient to remove the collagenase (Figure 4(d)).We further show no signifcant diferences in the total protein content using our collagenase protocol to digest the tendon allograft and a previously published pepsin digestion protocol (Figure 4(e)) [30,33].

DTM Promotes Cell Proliferation over Pepsin-Digested
Product.Bioactivity of the DTM was characterized in vitro using cell proliferation and gene expression analysis.Cellular proliferation was measured using the Presto Blue Assay for both tenocytes and adipose-derived stem cells (ADSCs).No signifcant diferences between treatment groups were found 48 hours after treatment in both tenocytes and ADSCs.However, 7 days following the initial seeding, DTMcoated plates had signifcantly more tenocytes and ADSCs compared to the pepsin-coated plates, respectively (Figures 2(a) and 2(b)).While DTM-coated plates were not signifcantly higher than the controls, the DTM-coated plates trended higher than the controls for both tenocytes and ADSCs at day 7. Still images taken at the time of cell plating and 48 hours later show that tenocytes have a varied cell morphology when cultured on diferent coatings.Specifcally, tenocytes were found to have a more native-like cell morphology on DTM-coated plates (Figure 2

DTM Promotes Tenocyte Maturation through Maintenance of TGFβ Bioactivity.
To characterize stem cell differentiation of ADSC in response to the DTM, RNA was harvested at day 2 and 7 following seeding.Expressions of the tenogenic markers Tenomodulin (Tnmd) and Scleraxis (Scx), and the chondrogenic transcription factor SRY-box transcription factor 9 (Sox9), were measured using qRT-PCR.Tnmd expression trended higher for ADSCs when cultured on DTM-coated plates as compared to pepsin groups and the control conditions (Figure 5(a)) at day 7. Scx expression also trended higher in the DTM-coated plates at day 7 as compared to both pepsin and control (Figure 5(b)).In addition to promoting tendon diferentiation, DTM promoted chondrogenic diferentiation as evident by an increase in Sox9 expression at day 7 than control (Figure 5(c)).Since soluble growth factors were not provided and TGFβ has been implicated in the development of the fbrocartilage enthesis [23,24], we next looked to see if Smad3, the downstream efector of TGFβ, was upregulated by the ADSCs following culture on DTM.DTM resulted in signifcantly more Smad3 expression at day 7 compared to control culture (Figure 5(d)).
Based on this increased expression of Smad3, we next looked to see whether the tendon allografts contained measurable levels of TGFβ at a protein level using Multiplex assays for TGFβ I, II, and III.Importantly, we found that adult tendon allografts do contain TGFβ and that there was no signifcant decrease in TGFβ I (Figure 6(a)) or TGFβ II (Figure 6(b)) expression following decellularization and enzymatic digestion.Interestingly, we did fnd signifcantly more TGFβ III (Figure 6(c)) in digested samples, DTM, and pepsin, compared to native tendon allografts.Additionally, no signifcant diferences were found between male and female donors in patella or Achilles tendons (Sup.Figure 1B).Further, we tested diferences in TGFβ activity   between male and female donors of the combined tendon samples.No signifcant diferences were found between sexes in TGFβI (Sup. Figure 2A), TGFβII (Sup. Figure 2B), or TGFβIII (Sup. Figure 2C).Additionally, no correlations were found between TGF-β I, II, or III activity and age of the donor (Sup.Figures 2A-2C).

DTM Augments Rotator Cuf Repair by Improving
Enthesis Biology.To determine if the in vitro bioactivity translated to a regenerative response, we completed an in vivo study in New Zealand White rabbits.Females were chosen due to the lack of sexual variability found within our preliminary data.Importantly, no signifcant diferences   8 Journal of Tissue Engineering and Regenerative Medicine were found between protein content of male and female Achilles or patella tendons (Supplemental Figure 1B) and additionally no diferences were found in TGFβI, II, and III content between male and female tendons (Supplemental Figures 2A-2C).Because many RCR failures are associated with chronic injuries, we utilized a rabbit model with a chronic rotator cuf tear (Figure 7(a)).In the groups that received DTM, the putty (1 mg rabbit DTM diluted in 3 mL PBS, rheological properties of rabbit DTM can be found in Supplemental Figure 3) was molded directly onto the greater tuberosity (Figure 7(b)).Repair then proceeded by securing the supraspinatus tendon to the greater tuberosity through 2 transosseous tunnels (Figure 7(c)).HBQ staining at the tendon-bone interface shows morphological diferences between DTM (Figure 7(f )) and the repair-only control group (Figure 7(e)).Images of contralateral shoulder enthesis reveal a cartilage transition, indicated by the red and blue colocalized staining in the matrix (Figure 7(d)).DTM also showed a cartilage transition at the junction (Figure 7(f )), yet little to no cartilage transition zone was present in the repair only (Figure 7(e)).Tese zones are labeled as FC = fbrocartilage zone, T = tidemark, and B = bone.Additionally, H&E staining reveals disoriented collagen fbrils near the enthesis within the RCR group alone (Figure 7(h), labeled with the arrow) as compared to both the contralateral shoulder and the DTM with repair groups.To further view the collagen orientation, we stained with Picrosirius Red showing more similarities in stain between the DTM group (Figure 7(l)) and the contralateral shoulder as compared to the repair only group (Figure 7(j)).Additional histological images can be found in Supplemental Figure 4.

Discussion
Tis study tested bioactivity of our engineered DTM putty with the long-term goal of promoting tendon regeneration during RCR.Because most rotator cuf tears result in diminished functionality of the shoulder joint, surgical repair remains the gold standard treatment for full thickness rotator cuf tears that appear to be amenable to repair.RCRs continue to have high clinical failure rates, with the majority of failures occurring at the tendon-bone interface due to the formation of a mechanically inferior scar tissue [5,12,34].
Given the importance of tendon healing for proper success of RCR, there remains a clinical need to fnd new, readily translatable approaches to reduce fbrosis and promote regeneration of a more native enthesis structure.Currently, there are few products designed to augment RCR, and those with clinical efcacy studies have reported minimal beneft [35].Recent tendon allograft studies suggest that modern surgical techniques yield functional improvements, yet MRI images illustrate poor tissue regeneration with tendon allograft and structural failure rate of 57% [10,36].Large clinical studies on RCR with various types of allograft are lacking, and there are no studies showing strong evidence of tissue regrowth into the allograft or regeneration of the enthesis structure [26].As an alternative to tendon allografts, dermal, small intestine submucosa and fascia lataderived human allografts have been tested for rotator cuf repair [37][38][39].While these allografts lack the standalone biomechanical properties of tendons [40], there are some clinical data suggesting efcacy [41][42][43][44][45]. Gupta et al. [42] performed a prospective observation of 24 patients who underwent a mini-open massive rotator cuf repair using a human dermal allograft at average follow-up of 3 years and demonstrated signifcant improvements in pain (VAS score; p � 0.0002), range of motion (forward fexion, external rotation, shoulder abduction; p ≤ 0.001), and patient reported outcomes (ASES, SF-12; p ≤ 0.3).At fnal follow-up, ultrasonographic evaluation revealed no evidence of complete tears, but 24% of patients only had a "partially intact" repair.In a more recent retrospective study in 12 patients (13 shoulders, 1 bilateral) that underwent revision rotator cuf repair with human acellular dermal allograft augmentation, Petri et al. found no complications, adverse reactions to the patch, or need for further surgery.Additionally, mean satisfaction was 9/10 (range, 2-10) and the total ASES score improved by 21.3 at mean follow-up of 2.5 years [46].Notably, only the functional component of the ASES score improved signifcantly (p < 0.05) and neither the total ASES score nor the pain component score improved by statistically signifcant amounts despite improving trends.
In this study, we have developed a processing technique to form a decellularized tendon allograft (DTM) putty with an optimized form factor to match modern arthroscopic surgical RCR techniques [32,47].Decellularization is considered a critical step in preventing a host infammatory response to the allograft and disease transmission [48,49].Unlike traditional tendon allograft processing techniques, we remove cellular content without detergents in an efort to maintain the protein bioactivity native within the tendon matrix, prevent harsh soft tissue matrix degradation, and reduce long processing time seen with detergents [27,32,33,50].
To improve the form factor of the allograft so that it was moldable for surgical application, we also added a collagenase digestion step.Conventional methods to circumvent allograft's poor form factor include enzymatic digestion or solubilization of the decellularized tissue [33,51,52].Traditional solubilization techniques use pepsin to preserve the ECM composition and structure.Pepsin is a proteolytic enzyme targeting telopeptide bonds of collagen giving rise to collagen fbril aggregates [52].Yet, pepsin necessitates an acidic base to facilitate the self-assembly of collagen peptides after being neutralized to physiologic pH, and this certainly alters the structural and functional integrity of the matrix [52].Farnebo et al. generated an injectable decellularized hydrogel derived from human fexor tendon using a pepsin solubilization technique to augment various tendonopathies and tendon injuries [33].When testing their product in a murine Achilles injury model, the groups that received the hydrogel resulted in higher ultimate failure load at earlier healing stages as compared to the control groups [53].While this shows promising results of an injectable-based decellularized tendon construct to augment Achilles tendon injuries, a more moldable and less viscous form would be more suitable to treat rotator cuf repairs.

Journal of Tissue Engineering and Regenerative Medicine
In testing DTM in vitro using tenocytes and ADSCs, we demonstrate greater cell proliferation in both cell types compared to pepsin-based tendon products.Gene analysis also suggested that DTM promoted the expression of Scx and Tnmd, tendon-specifc transcription factors required for tendon development, proliferation, and maturation [18,54,55].Furthermore, ADSCs cultured with DTM showed more Sox9 expression than all other groups tested, suggesting that DTM drives the Scx + /Sox9 + phenotype seen in tendon progenitor cells, the cell type required for postnatal tendon repair [21,22].
TGFβ has been reported to be critical for scarless tendon healing in neonatal mice through the recruitment and proliferation of Scx + /Sox9 + cells [13,15,23,24,56].In our study, we found that ADSCs cultured on DTM signifcantly upregulated Smad3 expression, suggesting that DTM promotes the Scx + /Sox9 + phenotype through retention of TGFβ.Consequently, we next measured the amount of TGFβ within the DTM samples and found that our processing technique did not lead to a signifcant loss of TGFβ when compared to the native tendon.While several studies have shown that the upregulation of TGFβI activity is associated with the age-related degenerative pathology in rotator cuf muscles, other studies display the necessary role TGFβI has during tendon enthesis repair [57].Importantly, TGFβI was found to be a key player throughout the early phases of rotator cuf healing in vivo coinciding with early ECM synthesis [58][59][60].In fact, it was determined to be the delayed upregulation of TGFβI (seen 8 weeks following injury) that was correlated with scarring [34,59,61].Additionally, various cell-ECM interactions such as substrate elasticity and proteolyticdegradation of ECM macromolecules infuence TGFβ levels [62][63][64][65][66][67].Tis suggests that DTM bioactivity may be due to the retention of the TGFβ family of proteins, which consequently recruit Scx + /Sox9 + tendon progenitor cells known to stimulate repair at the enthesis.
Preclinical animal studies are a critical step in demonstrating the efcacy of novel technologies for tendon repair.Due to limited applicability of rodent models for RCR in humans, large animal models, including rabbit and sheep, are typically required to explore efcacy and serve as more translationally relevant models [68][69][70][71].Importantly, chronic injury rabbit models show muscle atrophy, fatty infltration, and migration of the subscapularis tendon underneath a bony arch, characteristics comparable to massive tears in humans [69,72].For these reasons, we performed a pilot study in a rabbit model with a chronic rotator cuf injury, previously shown to replicate pathological changes consistent with a massive rotator cuf injury [73,74].Te rabbit model is also sufciently sized to enabled use of advanced arthroscopic surgical approaches used today to reduce re-tear rates [75][76][77][78][79].
Quality of the chronic rotator cuf injury repair in the DTM group was compared to standard surgical repair without biological augmentation after 8 weeks.When elucidating the pathogenesis of rotator cuf tears, Gigante et al. consistently found round cells characteristic of a chondrogenic lineage in rotator cuf tears, as compared to calcifed   Journal of Tissue Engineering and Regenerative Medicine fbrocartilage typically seen in non-torn tendon enthesis [80].Our results presented similar fndings as the RCR only group was shown to have cells phenotypically congruent with chondrocytes lacking the highly aligned calcifed fbrocartilage region of a normal enthesis.In contrast, the DTM repair group showed longitudinally oriented collagen fbers forming at the enthesis with a zone of calcifed cartilage at the bone-to-tendon interface that was more similar to the non-repaired contralateral shoulder control.Failure of RCR in chronic/massive tears has specifcally been associated with the formation of mechanically weak fbrovascular scars lacking the zone of calcifed cartilage [14][15][16].Tese preliminary in vivo data justify additional studies to further defne and quantify the four distinct regions of fbrocartilaginous entheses, especially the uncalcifed and calcifed zones of fbrocartilage.Tis study has some limitations.Te frst limitation was the small n value of the rabbit model enabling only preliminary data.We chose to use the rabbit model for these pilot study studies as it is a more clinically relevant method for rotator cuf repair; however, the expense and time of this model limited the n value.Additionally, these promising results from the pilot study performed necessitate more future in vivo studies to further confrm and expand upon these results, including biomechanical studies and quantitative histomorphometry.Another limitation concerning the in vivo study design was not testing the efcacy of pepsin-digested tendon in our rabbit model.Adding this as an experimental group to future studies in comparison to DTM would further expand upon the reported in vitro results.Limitations involving the in vitro studies include the diference in concentrations of well plate coatings between standard collagen-coated plates, DTM, and pepsin.Te last limitation was not testing rabbit DTM for metabolic activity of tenocytes and ADSCs in vitro.Testing the human DTM in an animal model will be considered in future steps although we also must consider possible xenographic immune reactions.
DTM represents an adaptation to allograft processing that can maintain bioactivity to promote better tissue regeneration.By utilizing tendon allografts as the basis for the DTM, we aimed to maintain the tissue-specifc bioactivity of the tendon.A similar approach has been used to develop an injectable decellularized tendon hydrogel; however, efcacy was only tested in Achilles injury models [27].While injectable systems may ofer advantages, such as non-surgical implantation, the disadvantages of injectable systems consist of highly digested matrices, low tendon material concentrations per injection, and the possibility of the product moving.However, DTM can be applied arthroscopically during RCR which circumvents the need for an injectablebased platform.
Complex strategies for engineered decellularized matrices have been proposed as a regenerative material, aiming to incorporate key parts of the tendon matrix and add back in cells or growth factors to promote cell migration or diferentiation [9,[81][82][83].Tese approaches may be very promising in the future but can be challenging and long to translate to the clinic.

Figure 1 :
Figure 1: (a) DNA content was measured following decellularization using the using the DTM processing technique or using standard detergent methods, like 1% SDS and 0.1% EDTA.All decellularization methods efectively removed DNA content as compared to treatment with PBS (p < 0.0001; n � 2 donors) and no signifcant diferences were found between decellularization treatments.Histological sections of (b) native tendon or (c) DTM decellularization stained with DAPI to identify cell nuclei, scale bar � 100 µm.An ordinary one-way ANOVA was conducted to determine statistical signifcance in DNA content between the treatment groups, F (5, 6) � 515.1, p < 0.0001, and all further signifcance gained from Tukey's multiple comparison testing is listed on graph (a).

Figure 2 :
Figure 2: Primary tenocytes and ADSCs were plated at 20,000 cells/well and proliferation was quantifed at 48 hours and 7 days (a, b) after plating, generating signifcantly diferent proliferation rates (n � 3-5 donors tested/group).DTM showed signifcantly more cell proliferation at day 7 than the pepsin groups in both tenocytes (p � 0.0001) and ADSCs (p < 0.0001).(c-j) Collagen coating, tissue-culture (TC) treated, DTM, and pepsin-coated plates were seeded with tenocytes and still images from live cell imaging were taken at 0 (c-f ) and 48 hours (g-j) where variance in tenocyte cell morphology can be viewed.A two-way ANOVA was conducted revealing a statistically signifcant interaction between days and treatment groups on cell number for ADSCs, F (4, 18) � 31.77,p < 0.0001, and for tenocytes, F (4, 16) � 5.150, p � 0.0073.Statistically signifcant results from Tukey's multiple comparison test are reported on graphs (a) and (b).Scale bars � 100 μm.

Figure 3 :
Figure 3: (a) Total protein content between native Achilles and patella tendons was analyzed and no signifcant diference was found between the tendon types (p � 0.7413; n � 10 donors).(b) Schematic diagram of Achilles and patella tendons dissected into equal thirds comprised of proximal (P), midcenter (M), and distal (D) regions.(c, d) Te patellar and Achilles tendons had no signifcance in total protein content between proximal, mid, and distal regions (p � 0.9893 and p � 0.368, respectively; n � 6 donors).Ordinary one-way ANOVA was conducted to determine statistical signifcance in total protein content and treatment groups, F (2, 33) � 0.934, p � 0.403, in patella tendon protein content and treatment groups, F (2, 15) � 0.011, p � 0.989, and in Achilles tendon protein content and treatment groups, F (2, 15) � 1.069, p � 0.368.

Figure 4 :
Figure 4: (a) DTM reconstituted and stretched to demonstrate the viscoelasticity of the DTM.(b) DTM reconstituted.(c) Oscillation stress sweep of human DTM with concentrations of 0, 1, 3, 5, and 7 g of lyophilized DTM to 1 mL of PBS resulted in all reconstitution concentrations tested to maintain an elastic modulus (n � 1 donor, 2-3 runs each).(d) Collagenase activity was measured to confrm successful removal of the added collagenase solution during enzymatic digestion.No signifcant diferences were observed in collagenase activity between native tendon and enzymatically digested and fltered samples (p � 0.922; n � 3-10 donors).(e) Te total protein of the samples was tested to confrm bioactivity was maintained (n � 4 donors) and no signifcant diferences were found between native, DTM, and pepsin samples.An ordinary one-way ANOVA was conducted to determine statistical signifcance in collagenase activity and treatment groups, F (4, 22) � 18.06, p < 0.0001, and in total protein content between groups, F (2, 9) � 5.282, p � 0.0304.Additional signifcance from Tukey's multiple comparison is listed on graphs (d) and (e).

Figure 7 :
Figure 7: Rotator cuf repair (RCR) surgery was performed in a rabbit model with a chronic rotator cuf tear (n � 4/group).(a) Initial rotator cuf tear of the supraspinatus; (b) DTM placed under the supraspinatus 6 weeks after the initial tear; (c) repair of the initial tear, sutureanchoring the supraspinatus to the greater tuberosity.8 weeks following the repair, shoulders were harvested, processed through histological analysis, and stained with (d-f ) HBQ, (g-i) H&E, and (j-l) Picrosirius Red.Images display the bone, fbrocartilage, and tendon tissues at the tendon enthesis in (d, g, j) contralateral shoulders, (e, h, k) repair only, and (f, i, l) DTM treatment with repair.T � tidemark; FC � fbrocartilaginous zone; B � bone; arrow in (h) shows collagen fber orientation.Scale bars represent 100 μm.
[31]Histological Analysis.Native Achilles and patella tendons, and processed DTM, were cut to a 1 cm × 1 cm square and cryo-embedded with Neg-50.Sections were cut to 6 µm and then stained with Fluroshield Mounting Media with DAPI to confrm decellularization (n = 3).Rabbit shoulders were fxed for 2 days in 4% paraformaldehyde and decalcifed for 4 weeks, shaking at 4 °C in 19% EDTA solution, changing solution every other day.Shoulders were cut down and processed for parafn embedding using a Tissue-Tek VIP 6 AI Vacuum Infltration Processor, set to a 1-hour cycle time.Once the bones were embedded in parafn, sections were cut to 6-8 µm.Hall Brundt's Quadruple (HBQ) staining and H&E staining of rabbit shoulders were done to visualize cartilage and bone.HBQ stain is a histological technique which can distinguish between cartilage (blue) and bone (red) and was frst published in 1986[30].HBQ uses direct red 81 stain to mark ossein and collagen fbers.Alcian blue stains structures involving connective tissue mucins and mucopolysaccharides[31]. VitroView ™ Picrosirius Red Stain Kit was used according to manufacturer's instructions to view the orientation of collagen fbrils, and was imaged under brightfeld microscopy.plicableandavailableresearch tissue.Animal sample size was determined by CSU cage availability and study funding.An overview of catalog number and vendor information where key biological resources were purchased can be found in Supplemental Table1.

Table 2 :
Summary of rheological properties of DTM, with the reconstitution concentration in column one, followed by complex modulus plateau (Pa), phase angle ( o ), and yield stress (Pa) (n � 1 donor, run 1 time).