Biomanufacturing organized collagen-based microfibers as a Tissue ENgineered Device (TEND) for tendon regeneration

Aligned TEND was produced by dissolving type I collagen (Collagen Solutions, Eden Prairie, MN) and UHMW PDLLA (Corbion, Amsterdam, Netherlands) in 100% DMSO (Gaylord Chemical, Slidell, LA) at a final concentration of 100 mg ml⁻¹ (30 mg ml⁻¹ collagen and 70 mg ml⁻¹ PDLLA) for 24 h. This polymer blend was electrospun onto a spoked-wheel collector in a vertical electrospinning setup. Evaporating the nonvolatile DMSO was accomplished by a hybrid form of electrospinning and pneumatospinning [39], providing constant pressure laminar airflow from a fan at the emitter directed towards the collector, and exhausting the air from directly above the collector. The surface speed of the drum was 18 m s⁻¹. The distance between the needle and the collecting drum was 130 mm. A 25-gauge needle was used, and the solution flow rate was set to 1 ml hr⁻¹. Humidity was maintained between 45%–50% and temperature was between 23 °C–26 °C. Upon completion of the electrospinning process, TEND was removed and placed under vacuum for 24 h at ambient temperature to aid with removal of any residual DMSO. Throughout this manuscript term 'TEND' has been used to address all electrospun grafts in general. Term 'as-electrospun TEND' is used when this condition is compared to the annealed TEND as explained in section 2.2.

Most electrospun materials significantly shrink due to thermally induced relaxation of stretched amorphous chains [40]. However, thermal annealing of many polymers [41–44] has led to improvements in their mechanical stability by changing microstructural properties and their degree of crystallinity. Here, as-electrospun TEND was gripped in aluminum frames and held under tension during post-processing. The framed sheets were annealed under vacuum at various temperatures and durations for optimization. TEND annealed at 65 °C for 18 h demonstrated highest degree of stability compared to all the temperatures and durations tested, therefore, for the remainder of experiments all TEND were annealed under these conditions. Throughout this manuscript the electrospun grafts that have been annealed are referred to as 'annealed TEND'.

SEM was performed at Jefferson Labs (Newport News, VA) using a JEOL JSM-6060 LV microscope (JEOL Ltd., Tokyo, Japan) with a 20 kV beam intensity. The SEM images were analyzed with ImageJ software (NIH Shareware, Bethesda, MD) to determine fiber diameter and fiber alignment of annealed and as-electrospun TEND at day 0 and after a 14-day incubation in Dulbecco's modified eagle medium (DMEM) at 37 °C with 5% CO₂.

SDS-PAGE was comparatively used to identify the alpha I and II chains present in monomeric triple helical collagen feedstock (Collagen Solutions), and in annealed and as-electrospun TEND. Samples were run on NuPAGE 3%–8% tris-acetate gels at 150kV on PowerEase 300 W. The gel was removed and stained with SimplyBlue gel stain for 1 h and then rinsed with deionized water for 1 h. All items were from Thermo Fisher Scientific.

FTIR was performed on Platinum ATR (Brucker, Billercia, MA) at Old Dominion University (ODU) (Norfolk, VA) to confirm the presence of the three amide bonds characteristic of type I collagen at 1235, 1560, and 1650 nm wavelengths. Bond peaks of electrospun and annealed TEND were compared to collagen and PDLLA feedstocks.

Mass spectrometry was used to analyze the presence of DMSO in TEND after 0, 2, 4, 6, and 24 h under vacuum. After vacuum, approximately 20 mg of test material was suspended in 3 ml of Dimethylacetamide and heated to 60 °C with agitation for 24 h. Supernatants were collected for GCMS analysis. Replicate extracts were prepared for each sample. A second extraction was conducted to confirm complete recovery of residual DMSO. The extracts were run on Agilent 7890 A GC. The GCMS testing was performed by InVision Biomedical Group, Inc. (Irvine, CA).

TGA analysis was performed to assess the presence of residual DMSO within TEND. TGA data of as-electrospun TEND samples placed under vacuum for 0 and 24 h was obtained from a TGA Q5000 (TA instruments, New Castle, DE) at ODU. Data was collected at a rate of 5 °C min⁻¹ from 21 °C to 300 °C.

Mercury intrusion porosimetry was performed at Quantachrome Instruments (subsidiary of Anton Paar, Boynton Beach, FL) on triplicate samples of as-electrospun and annealed TEND on a PoreMaster 60, with analyses performed blinded to sample identity. Gas sorption was also performed at Quantachrome on triplicate samples of as-electrospun or annealed TEND on an AutoSorb IQ, with analyses performed blinded to sample identity, including calculation of mean Brunauer–Emmett–Teller (BET) surface area.

TEND used for these studies were cut into the required sizes and inserted into Tyvek pouches (4MD Medical Solutions, Lakewood, NJ). Tyvek pouches were sealed with a heat sealer and sent for electron beam sterilization (Steri-Tek, Fremont, CA) using a 20 ± 2kGy target dose. Mechanical testing and chemical characterization of TEND were performed before e-beam sterilization.

E-beam sterilized as-electrospun and annealed TEND were incubated in DMEM with 1% antibiotic-antimycotic (ABAM) (Thermo Fisher Scientific, San Diego, CA) at 37 °C with 5% CO₂ for seven days. After seven days, their mechanical properties were tested by uniaxial tensioning in the direction of aligned fibers at room temperature using an MTS Criterion Model 42 (Eden Prairie, MN) at a rate of 1 mm s⁻¹. As-electrospun and annealed TEND soaked in Dulbecco's phosphate buffered saline (DPBS) (Fisher Scientific, Hampton, NH) for 30 min were used as Day 0 controls. The dimensions of the samples at day 0 and day 7 were used to calculate the percentage of material shrinkage. The fiber diameters assessed using SEM images were used to calculate percentage of material swelling.

Human blood with anticoagulant heparin and citrate dextrose (ACD-A) was purchased from Biological Specialty Corporation (Colmar, PA). A calcium chloride (CaCl₂) (Fisher Scientific) concentration of 1.25% was found required to initiate coagulation in clotting blood supplemented with ACD-A. Annealed TEND samples (40 × 40 × 1 mm) were immersed in clotting blood (ACD-A + CaCl₂) and heparanized, non-clotting blood. Soaked samples were weighed in intervals of 5 min to calculate the amount of blood absorbed. Volume of absorbed blood was calculated by using density of 1 ml of blood. Dimensional changes after blood hydration was calculated by snap freezing the samples in liquid nitrogen and measuring the size using calipers (n = 3). Samples immersed in blood as above and in a fresh draw from a rabbit ear vein were fixed in 2.5% glutaraldehyde in 0.1 M Sodium cacodylate buffer, pH 7.4 (Electron Microscopy Sciences, Hatfield, PA) at 4 °C. Fixed samples were washed with 3X phosphate buffered saline (PBS) (Thermo Fisher Scientific) for ten minutes, placed in 100% hexamethyldisilazane (HMDS) (Electron Microscopy Sciences, Hatfield, PA) for 3 min, and then air dried. Samples were further imaged by SEM.

Human PRP with anticoagulant citrate dextrose (ACD-A) (ZenBio, Research Triangle Park, NC) was stored at 4 °C for 30 min, then calcium chloride (CaCl₂₎ (Fisher Scientific) was added to reach a final concentration of 22 mM. The PRP solution was mixed and placed at 37 °C for 1 h to activate. After clotting, the lysate was collected for use on annealed TEND. Annealed TEND (10 × 10 mm) (n = 4) and size-matched rabbit Achilles tendon (n = 4) were soaked in 1 ml of PRP lysate for 6 min followed by placement in 600 µl of modified eagle medium (MEM) (Fisher Scientific) with 2% ABAM). Negative controls consisted of annealed TEND soaked for 6 min in MEM with 2% ABAM only. Samples were incubated at 37 °C with 5% CO₂. After 30 min, 4, 8, 24 h, and 3, 7 and 14 d, MEM was collected from each well and stored at −80 °C. MEM was replenished after each time point.

TENDS's binding and release of growth factors typically found in high concentrations in PRP was assessed using ELISA's specific for TGF-β1 and PDGF-BB (R&D Systems, Minneapolis, MN and RayBiotech, Norcross, GA). The elution kinetics of TGF- β1 and PDGF-BB from the electrospun grafts and native tissue was assessed per the manufacturer's instructions at the timepoints per section 2.12.

Two sets of annealed TEND were cut into 10 × 30 mm (n = 6 per group). One set of samples were soaked in PRP for 10 min. PRP soaked TEND and a second set of non-coated TEND were placed in grips under static tension and remained in culture at 37 °C and 5% CO₂ for up to seven days. The samples were removed from the grips after 3 and 7 d in culture and their mechanical properties were tested at room temperature by uniaxial tensioning in the direction of aligned fibers using an MTS Criterion Model 42 at a rate of 1 mm s⁻¹. Annealed TEND soaked in DPBS (Fisher Scientific) for 30 min were used as Day 0 controls.

Human tenocytes (ZenBio) were cultured in tenocyte culture medium (ZenBio). Human bone marrow-derived MSCs (RoosterBio^®, Ballenger Creek, MD) were cultured and maintained in serum-free, xeno-free media (RoosterBio^®). All cell cultures were maintained under physiological conditions at 37 °C and 5% CO₂.

To assess human tenocyte cell proliferation on TEND, one set of each as-electrospun or annealed gripped TEND (10 × 30 mm) (n = 6) and one set of each as-electrospun or annealed loose TEND (10 × 30 mm) (n = 3) were seeded with 2.5 × 10⁴ human tenocytes per TEND to allow sufficient surface area for cell growth over the course of two weeks. Cellular activity was assessed after 1, 7 and 14 d in culture. The alamarBlue^® metabolic activity assay (Bio-Rad) was used per the manufacturer's protocol to test cellular viability and proliferation. Cell infiltration and morphology was assessed over 14 d in culture. One set of each as-electrospun or annealed gripped TEND (n = 3) and one set of each as-electrospun or annealed loose TEND (n = 3) were seeded with 1 × 10⁵ cells/construct. Cellular infiltration was assessed after 1, 7 and 14 d in culture. Cells were fixed in 4% paraformaldehyde and stained with 4',6-diamidino-2-phenylindole (DAPI) (Fisher Scientific) nucleic stain. Cellular infiltration from the surface of TEND was measured with a Zeiss Axio Observer Z1 confocal microscope (Zeiss, Oberkochen, Germany). Five fields of view per TEND were investigated. At each field of view, multiple 5 μm thick z-scan slices were captured, and the total depth of cell infiltration was calculated based on the number of cell-containing slices captured. The total depth was averaged for the five fields of view. To visualize cell morphology and elongation, all TEND cultured with human tenocytes were fixed in 4% paraformaldehyde and stained for nuclei and actin filaments using DAPI nucleic stain and Alexa Fluor^® 594 phalloidin (Thermo Fisher Scientific), respectively. The stained samples were imaged using a Zeiss Axio Observer Z1 confocal microscope. Percentage of cell elongation was determined based on the average calculated nuclei aspect ratio by measuring the length and width of twenty cell nuclei using ImageJ FIJI (NIH Shareware). Measurements were performed using confocal images taken at 40X magnification.

To compare mechanical stability of acellular and cellularized TEND, one set of each (n = 12) was placed in custom grips and held under static tension (gripped) to prevent shrinkage and promote further fiber alignment. A second set of annealed TEND (n = 12) was placed in ultra-low cell binding culture plates without tension (loose). Half of each set of TEND, gripped (n = 6) or loose (n = 6), were seeded with 1 × 10⁵ human tenocytes per side for a total of 2 × 10⁵ cells per TEND. All samples remained in culture for 7 and 14 d. At each time point, the samples were removed from the wells and pulled to failure under uniaxial tension in the direction of the aligned fibers. Annealed TEND at day 0 (controls) were hydrated in DPBS for 30 min prior to testing. All samples, cellularized and acellular, were mechanically tested at room temperature using an MTS Criterion Model 42 (Eden Prairie, MN) at a rate of 1 mm s⁻¹. Thickness was measured with a Mitutoyo 547–526 S (Takatsu-ku, Kawasaki, Japan) thickness gauge.

Annealed TEND (n = 6) were pre-coated with human PRP for 10 min. Clinical grade mesenchymal stem cells derived from human bone marrow (RoosterBio^®) were seeded on the PRP coated TEND at 5 × 10⁵ cells/TEND. One half of the constructs (n = 3) were placed in a custom designed cyclic-loading bioreactor. The spring-driven load on the bioreactor had a spring constant of 0.125 N mm⁻¹ and was cycled between 0 and 8 mm resulting in a 0–1 N sinusoidal wave. The bioreactor chamber contains 316 stainless steel, polyether ether ketone (PEEK) and medical grade silicone. The polycarbonate window provided two ports for gas exchange. A second set of cell seeded constructs (n = 3) were placed in static grips and kept under static tension. Cultures were maintained with defined media for three days prior to qPCR analysis.

RNA extraction was performed by pooling n = 2 or 3 individual TEND samples and lysing the cells within TEND directly in complete lysis buffer from the RNEASY Plus mini prep kit (Qiagen, Valencia, CA). TEND samples were cut into small pieces and then vortexed vigorously for 10 min. RNA was extracted following the manufacturer's protocol. The amount of RNA extracted was measured using a NanoDrop spectrophotometer. Up to 750 ng of total RNA was converted to cDNA using the protocol described in the SuperScript™ III First-Strand Synthesis SuperMix kit (Life Technologies, Grand Island, NY). 40–50ng of cDNA was used in qPCR reactions following the manufacturer's protocol for the SoFast^™ Evagreen^® Supermix (Bio-Rad, Hercules, CA) and primers specific to the target human genes were obtained from published references [45–50]. Reactions were run in triplicates on the CFX Connect^™ Real-Time PCR Detection System (Bio-Rad) on 96 well plates, using 39 cycles with an annealing temperature of 56 °C. A single amplicon product was confirmed by the detection of a single peak on the melt curve. mRNA transcript levels were normalized to corresponding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) values. Results were consolidated and analyzed using the instrument's supporting software (CFX Maestro Software, BioRad).

All surgical procedures were conducted according to a protocol approved by Institutional Animal Care and Use Committee (IACUC), Old Dominion University. Per ISO 10 993–6, n = 6 grafts of TEND and allograft dermis (Wright Medical, Memphis, TN) were implanted for 2, 4, 8 and 16-weeks in a pool of male and female Sprague Dawley rats. Rats were anesthetized with isoflurane inhalation. Flanks were shaved, and Nair depilatory cream was applied to remove hair from the surgical site. Incisions were made dorsally in the flank area and a hemostat was used to create a pocket for implants. Once grafts were implanted in the pocket, the incision was closed using suture. After the designated time points, the rats were humanely euthanized for tissue collection.

All surgical procedures were conducted according to a protocol developed based on studies by Van Kampen et al [51] and approved by Institutional Animal Care and Use Committee (IACUC), Eastern Virginia Medical School. TEND grafts were implanted for 4, 16 and 52-weeks in the Achilles tendon of male New Zealand White Rabbits and compared with sham (suture alone). Rabbits were administered with Acepromazine and anesthesia induced using isoflurane. Clippers and Nair depilatory cream were used to remove hair from hind legs. Surgical sites were cleaned and aseptically prepared with alternating passes of betadine and 70% alcohol. The calcaneal tendon was exposed through paratendineal incision of the cutis, subcutis and fascia. Using a number 10 scalpel blade, three full thickness incisions (1 incision per bundle) approximately 7 mm long were made in the coronal plane of the calcaneal-Achilles tendon, midsubstance between the calcaneus and the gastrocnemius. Annealed TEND was circumferentially placed around the Achilles tendon defects (similar to the implantation in the cadaver model shown in figure 12) and sutured into place using 4–0 Mersilene^®. The peritenon was closed using 4–0 Vicryl^® suture. The incision site was closed with a 4–0 Vicryl^® running suture. For sham, sutures were placed in identical pattern as the graft using 4–0 Mersilene^® and 4–0 Vicryl^® to close the peritenon. After surgery, antibiotic was placed on the incisions and the operated legs wrapped with surgical gauze and cotton roll. Vet wrap was used to wrap the leg and hold it in place. Surgical gauze, cotton roll and Vet wrap were removed after 24 h. Animals were fed using a food hopper from inside the cage after surgery. The rabbits were not allowed outside of their cages for 14 d post-op in order to restrict their movements to ensure proper healing of the defect. At the endpoint, the animals were euthanized by deep anesthesia with isoflurane inhalation followed by barbiturate overdose (1cc per 10 pounds) with confirmation of death with a bilateral thoracotomy.

Harvested graft explants were fixed in 4% paraformaldehyde (Alfa Aesar, Haverhill, MA) for 24 h then transferred to PBS. Samples were sectioned to 5 μm thickness and serial sections were stained with Masson's trichrome and H&E at IDEXX (West Sacramento, CA). QuPath, an open source software, was used to quantify the number of cells on the histology sections. Three H&E slides of each graft types were chosen, and images taken using 10X magnification. A representative image of the graft from each slide was selected to run on QuPath. Polarized light microscopy was used to image newly produced collagen within TEND implants and sham controls. The alignment of the deposited collagen was assessed using Directionality function of ImageJ software.

Cadaveric implantation was performed in the prone position through a posterior transverse 3-centimeter incision at a level 4–6 centimeters from the superior margin of the calcaneus. The Deep Fascia and Paratenon were incised and maintained for later closure. An irregular cut was made full thickness through the Achilles tendon to mimic a clinical rupture. A minimally invasive repair technique was performed with a suture passer using suture tape passed along each side of the proximal tendon to the level of the rupture. The suture was then passed through the distal tendon rupture using a suture retriever, passed through a transverse bone tunnel in the calcaneus, tensioned to bring the ruptured ends together and tied with a surgeon's knot. TEND was then properly oriented and passed via the plastic inserter under and around the Achilles tendon repair site. It was fixated with standard 3–0 Vicryl suture to the host tendon. A 22-gauge needle was used to make multiple passes through TEND and into the Achilles tendon to mimic the use of PRP injection augmentation. The Paratenon and Deep Fascia was closed with 3–0 Vicryl. Skin was sutured with 3–0 Nylon. The ankle was then taken through a range of motion to check the integrity of the repair with TEND in place. Inspection of the repair and TEND was then performed by careful dissection of the surrounding skin and subcutaneous tissue.

Data analysis was performed on Prism 8 software. All parameters are expressed as mean ± S.E.M. Two-way ANOVA followed by the post-hoc Tukey's Multiple Comparison Test and Unpaired t tests were used to assess the differences in mechanical properties and fiber diameters. The Kolmogorov-Smirnov test was used to assess the differences in TEND fiber alignment and newly formed collagen alignment in vivo. Paired t test was used to assess the differences in PRP release kinetics. The chi-square test was used to assess the differences in frequency distribution of cellular alignment. Two-way ANOVA followed by the post-hoc Tukey's Multiple Comparison Test was used to assess the differences in in vitro cell elongation, cell attachment, proliferation, and infiltration, mechanical properties of the cellularized and acellular TEND, and cell quantification of rat subcutaneous implants. An unpaired t-test and ordinary one-way ANOVA followed by the post-hoc Dunnett's Multiple Comparison Test was used to assess the differences in gene expression. A priori, p values below 0.05 were defined as significant in all studies.

Hybrid pneumatospinning—electrospinning setup allowed manufacturing of highly aligned collagen:PDLLA fibrous grafts from 100% DMSO. The resulting as-electrospun TEND exhibit an average peak stress of 13.6 ± 0.26 MPa, modulus of 152.2 ± 15.85 MPa, and could withstand 8.4 ± 1.77 N load. In order to improve the porosity and void fraction of TEND, as-electrospun TEND were heated above their glass transition temperature for annealing. As demonstrated in figures 2(A) and (B), the annealing process caused TEND to expand in thickness, hence increasing the void fraction within the construct. The physical and mechanical properties of annealed and as-electrospun TEND where tested in culture and compared to assess the effects of annealing. The structure and alignment of both as-electrospun and annealed TEND at day 0 and 14 d in culture are shown in figures 2(C)–(F). Annealing under tension resulted in significantly smaller fiber diameters (p < 0.01) and higher degree of fiber alignment (p < 0.05) compared to as-electrospun TEND at day 0 or after 14 d in culture (figures 2(G) and (H)). The higher degree of loss of fiber alignment resulting in formation of disorganized fibers in as-electrospun TEND after remaining in culture is due to shrinkage of TEND. It was demonstrated that annealing resulted in significantly smaller amount of TEND shrinkage in culture compared to as-electrospun TEND (figure 2(I)). Uniaxial tensile testing of TEND revealed that there was no significant difference in the peak stress and modulus of annealed and as-electrospun TEND at day 0 (figures 2(J) and (K)). Although both annealed and as-electrospun TEND exhibited a significant decrease in their peak stress and modulus after 7 d in culture, it was shown that annealed TEND retained significantly higher peak stress (2X higher) and modulus (11x higher) compared to as-electrospun TEND (figures 2(J) and (K)). Furthermore, at day 7 there was no significant decrease in the load bearing capability of TEND compared to Day 0 (figure 2(L)). Day 0 samples used for mechanical testing were pre e-beam sterilization, however TEND tested post sterilization exhibited no significant difference in its mechanical properties (Data not shown). Since annealing significantly increased the thickness of as-electrospun TEND, we tested if the internal void fraction of TEND was altered pre- vs. post-processing. Mercury intrusion porosimetry analysis showed that annealing process increases the average pore size of aligned fibers from 7.1 µm to 100.7 µm (figure 2(M)). Gas sorption showed that annealed TEND's 'pore' structure throughout the graft was large slit-shaped, per the adsorption-desorption hysteresis curves (figure 2(N)). Additionally, annealing increased the graft surface area significantly, as validated by gas adsorption methods (figure 2(O)).

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SDS-PAGE gel electrophoresis was used to examine the impact of electrospinning and heat on collagen protein in TEND. Collagen starting material and annealed TEND were dissolved in acetic acid and run on a gel (figure 3(A)). Distinct bands visible around 238kD and 117kD confirm the presence of alpha and beta regions, respectively within TEND. Moreover, lack of smearing or laddering below 117kD showed collagen did not molecularly breakdown due to manufacturing. The difference in band intensity between collagen raw material and annealed TEND is due to different amounts of collagen loaded as the raw material is 100% collagen and the same amount of TEND in weight only contains 30% collagen. The lower band intensity due to smaller amount of collagen is not suggestive of denaturation into gelatin, as alpha, beta and gamma chains are all present in TEND as in the starting material, while there is no laddering effect that migrates faster than the alpha chain. TEND fiber crystallinity assessed by XRD showed collagen feedstock with a broad peak at approximately 20 degrees, as seen in as-electrospun and annealed TEND with a sharper peak within the same region (figure 3(B)), indicating that TEND maintained collagen crystallinity post-processing. FTIR results indicate that as-electrospun and annealed TEND had amide I (~1650 cm-1) and amide II (~1560 cm-1) which are the fingerprints of collagen, as well as amide A (~3285 cm-1) and amide B (~2917 cm-1) (figure 3(C)). The presence of amides I, II, A, and B confirmed collagen presence in TEND post-annealing. TGA was run on as-electrospun TEND before and after vacuuming. After 24 h under vacuum, 95% of the weight lost is closer to 250 °C rather than with negligible loss at 189 °C, the boiling point for DMSO (figure 3(D)), indicating DMSO was removed during the manufacturing process. Removal of DMSO was further confirmed by mass spectrometer. GCMS results indicate that after 24 h of vacuum, average percent by weight of DMSO in TEND dropped from 1.74% to 1.48% (figure 3(E)) which is > 1/1000th of the 50 mg day⁻¹ allowed in an implant per USP < 467 > . SDS-PAGE of TEND post e-beam sterilization also showed the presence of collagen α chains (Data not shown).

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As patient blood is commonly drawn for preconditioning surgical implants and may be either heparinized or non-heparinized, the blood absorption capability of TEND was measured by soaking in clotting blood and comparing it to heparinized blood. After 35 min on average, TEND soaked in clotting blood absorbed more than twice the volume (3.95 ± 0.43 ml) compared to TEND soaked with heparinized blood (1.93 ± 0.12 ml) (figure 4(A)). After soaking in blood for 45 min, the length, width and thickness of each sample was measured. The percent change in volume between the heparinized and clotting blood-soaked TEND were found to be insignificant (p > 0.05). SEM images of the human blood-soaked TEND showed red blood cell and platelet attachment on the samples (figure 4(B)).

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The PRP release kinetics results demonstrated that annealed TEND sequester and controllably release PRP over 14 d, as measured by release of PDGF-BB and TGFβ1 (figures 5(A) and (B)). Overall, TEND binds and releases significantly higher amounts of PRP-derived growth factors compared to native tendon (figures 5(A) and (B)). Moreover, TEND coated in PRP and incubated under static tension exhibit improved mechanical stability and tensile strength in culture demonstrated by significantly higher peak stress and modulus (figures 5(C) and (D)) as related to changes in graft thickness in PRP. There were no significant differences observed in peak load over time (figure 5(E)), showing high graft stability upon use. Additionally, PRP-coated TEND exhibited lower strain at break than non-coated TEND, although this difference was significant only at the 3-day time point. The lower strain at break within the PRP-coated TEND is representative of the increase in modulus (**p < 0.01) (figure 5(F)).

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Cellular morphology and elongation of human tenocytes on as-electrospun or annealed TEND were assessed. The direction of actin filaments (red), shown in the confocal images (figures 6(A)-a and (A)-b) demonstrated cell elongation on the gripped annealed and gripped as-electrospun TEND along the direction of the fibers (figures 6(A)-e and (A)-f). The random morphology of actin filaments in the cells on loose TEND (figures 6(A)-c and (A)-d) demonstrated the loss of fiber alignment within these constructs. This random morphology is confirmed by SEM images of the fibers shown in figures 6(A)-g and h. Cell aspect ratio measurements demonstrated that overall, human tenocytes had a higher percentage of elongation within the gripped TEND compared to the loose TEND (p < 0.01) (figure 6(B)). This is due to the higher degree of fiber alignment and tension within the gripped TEND (figure 6(C)), resembling gripping in surgical graft fixation. On the contrary, within loose TEND there was significant loss of fiber alignment due to the absence of tension (figure 6(C)). Although loss of fiber alignment was observed within loose samples, it was shown that annealed TEND was still significantly more aligned than the as-electrospun TEND after 14 d in culture (figure 6(C)) highlighting the importance of the annealing process.

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As annealing led to improved tensile, material and 'porosity' properties of TEND, only annealed TEND was used for the remaining in vitro studies. Gripped and loose TEND were seeded with human tenocytes to assess the effects of cells on their mechanical strength and physical properties. After 7 and 14 d in culture, the samples were pulled to failure in the direction of aligned fibers. Overall, after 7 or 14 d, all TEND in culture lost a significant amount of strength and modulus compared to the control groups (p < 0.05). However, gripped TEND was significantly stronger and stiffer than loose TEND regardless of the presence or absence of cells (p < 0.05) (figures 7(A) and (B)). TEND without cells was significantly stronger than TEND with cells when gripped for 7 d in culture (p < 0.05) (figure 7(A)). However, after 14 d, the mechanical strength of TEND was maintained in the presence of cells (p < 0.05) (figure 7(A)). Although TEND in culture showed lower peak stress and modulus, there were no significant changes in the amount of load they could withstand (p > 0.05) (figure 7(C)). As load remained consistent and the dimensions of TEND changed while in culture, all effects on the calculated mechanical properties can be attributed to the swollen cross section leading to an increased cross-sectional area. After 14 d in culture, loose TEND had significantly higher strain at break than the other conditions tested (p < 0.05). Overall, all TEND in culture had significantly higher strain at break than controls (p < 0.05) (figure 7(D)).

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TEND was coated with PRP to simulate prospective clinical use as a biological delivery vehicle with patient-derived PRP or other blood-based products. Clinical grade human MSCs grown on PRP-coated TEND in both static and bioreactor cultures for 3 d showed statistically significant upregulation of collagen type I, collagen type III, and tenascin C genes compared to the same cells cultured on tissue culture plates alone or supplemented with BMP-12 to induce tenogenic differentiation (figure 8). This suggests upregulation of tendon-like matrix synthesis and induction of tenogenic differentiation from cultured stem cells on TEND. Ki67 downregulation in stem cells grown on PRP-coated TEND further indicated that stem cells proliferation was reduced suggesting exiting of the cell-cycle due to lineage commitment.

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As-electrospun and annealed TEND were implanted subcutaneously in rats to assess biocompatibility, cellular infiltration and remodeling between the groups with different porosities/bulk densities yet otherwise identical grafts. At 2, 4, 8, and 16 weeks TEND and decellularized human dermis were explanted, sectioned and stained with hematoxylin & eosin (H&E). Histology results and analyses of cell numbers within grafts at different time points showed that a higher number of cells infiltrated TEND in annealed groups compared to as-electrospun TEND (figure 9). Annealed TEND showed significantly higher cellular infiltration after 2 weeks (figure 9(B)). Allograft dermis exhibited an intense early inflammatory response at 2 weeks (figure 9(C)) with primarily mononuclear cells. Annealed TEND showed increased cellularization after 4 and 8 weeks (figures 9(E) and (H)) whereas allograft dermis progressed to fibrotic encapsulation shown at 8 weeks (figure 9(I)). Annealed TEND showed histological evidence of graft remodeling into dense host connective tissue by 16 weeks (figure 9(K)) while persistent fibrotic encapsulation was still present around allograft dermis (figure 9(L)).

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Annealed TEND was further tested in a clinically relevant tendon injury model by implantation around a Achilles tendon defect in rabbits (figure 10) to assess cellular infiltration, remodeling, and regeneration. TENDs were explanted at 16 and 52 weeks and stained with H&E and Masson's trichrome. Histology images of suture only (sham) explants showed inflammatory cells at 16 weeks (figure 11(A)) and disorganized collagen formation (figures 11(B) and (C)). On the contrary, it was shown that annealed TEND was cellularized and started remodeling by 16 weeks (figures 11(D)–(F)). By 52 weeks, histology images indicate that TEND had fully remodeled and new collagen was formed. It was shown that the newly formed collagen in the sham group was randomly orientated (figures 11(G)–(I)) whereas highly organized, aligned collagen was formed within TEND (figures 11(J)–(L)). Collagen alignment was assessed by directionality function of ImageJ and it was shown that collagen formed in the presence of TEND, had significantly higher degree of alignment when compared to suture-only sham. This higher degree of alignment is denoted by the narrower width and higher peak of the histogram shown in figure 11(M).

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Current surgical repair of the ruptured Achilles tendon is performed through either an open approach (10–14 centimeter incision) or more commonly through a minimally invasive (3–4 centimeter incision) surgical technique in order to avoid the common postoperative wound complications. There are two commonly used grafts in the repair of Achilles tendons. Human decellularized cadaveric dermis and various forms of human preserved amniotic placental membrane. Both of these grafts have problems with implantation or fixation to the host tendon. Cadaver surgical implantation of TEND was performed with a minimally invasive technique that allowed easy graft orientation (colored fibers, figure 12) and delivery with its plastic inserter. TEND showed excellent handleability and suture fixation to the native tendon. These inherent characteristics of TEND should aid surgeons in clinical practice.

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