HAND SURGERY
In Vitro Characteristics of Porcine Tendon Hydrogel for Tendon Regeneration Christopher S. Crowe, BS,*† Grace Chiou, MD,*† Rory McGoldrick, MBBS, MBA,*† Kenneth Hui, BA,*† Hung Pham, BS,*† Emily Hollenbeck, BS,*‡ and James Chang, MD*† Purpose: Previous work has characterized the development of a human tendon hydrogel capable of improving mechanical strength after tendon injury. Animal tendon hydrogel has not yet been described, but would prove beneficial due to the cost and ethical concerns associated with the use of human cadaveric tendon. This study details the manufacture and assesses the biocompatibility of porcine tendon hydrogel seeded with human adipoderived stem cells (ASCs). Materials and Methods: Porcine tendon was dissected from surrounding connective and muscle tissue and decellularized via 0.2% sodium dodecyl sulfate and 0.2% sodium dodecyl sulfate/ethylenediaminetetraacetic acid wash solutions before lyophilization. Tendon was milled and reconstituted by previously described methods. Decellularization was confirmed by hematoxylin-eosin staining, SYTO Green 11 nucleic acid dye, and DNeasy assay. The protein composition of milled tendon matrix before and after digestion was identified by mass spectrometry. Rheological properties were determined using an ARG2 rheometer. Biocompatibility was assessed by live/dead assay. The proliferation of human ASCs seeded in porcine and human hydrogel was measured by MTS assay. All experimental conditions were performed in triplicate. Results: Decellularization of porcine tendon was successful. Mass spectrometry showed that collagen composes one third of milled porcine tendon before and after pepsin digestion. Rheology demonstrated that porcine hydrogel maintains a fluid consistency over a range of temperatures, unlike human hydrogel, which tends to solidify. Live/dead staining revealed that human ASCs survive in hydrogel 7 days after seeding and retain spindle-like morphology. MTS assay at day 3 and day 5 showed that human ASC proliferation was marginally greater in human hydrogel. Conclusions: After reconstitution and digestion, porcine hydrogel was capable of supporting growth of human ASCs. The minimal difference in proliferative capacity suggests that porcine tendon hydrogel may be an effective and viable alternative to human hydrogel for the enhancement of tendon healing. Key Words: tissue engineering, porcine tendon, tendon repair, xenograft (Ann Plast Surg 2016;77: 47–53)
T
endon healing represents a complex process characterized by both intrinsic and extrinsic mechanisms.1 After injury, tenocytes are capable of proliferating and migrating to the site of repair. In addition, healing may also be driven by the ingrowth of other local cells and vascularization from adjacent tissue. The biology of tendon healing becomes clinically relevant when viewed as the basis of functional outcomes. With repetitive stress, such as the case with tendinopathy, the balance between extracellular matrix (ECM) synthesis and degradation can be tipped.2,3 Additionally, disorganized synthesis of collagen fibrils Received July 28, 2014, and accepted for publication, after revision, September 1, 2014. From the *Division of Plastic and Reconstructive Surgery, Stanford University Medical Center, Stanford; †Section of Plastic Surgery, VA Palo Alto Health Care System, Palo Alto; and ‡Department of Chemical Engineering, Stanford University, Stanford, CA. Conflicts of interest and sources of funding: The laboratory is funded by several Federal grants including the VA RR&D Merit Review program. Reprints: James Chang, MD, Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University Medical Center, Stanford, CA. E-mail: [email protected]. Copyright © 2014 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0148-7043/16/7701–0047 DOI: 10.1097/SAP.0000000000000361
may be noted microscopically,4,5 translating into diminished tensile strength of the tissue. Understanding how tendon healing proceeds, and more importantly how it fails, provides considerable insight into the challenges that new interventions must mitigate. Because tendon repair follows a slow course that is complicated by many factors, a variety of methods to stimulate and aid healing have been attempted. These include injections of whole blood,6 plateletrich plasma,7–9 multipotent stem cells,10–12 and combinations of growth factors and hormones.6,13,14 Some attention has been paid to injectable hydrogels—particles dispersed within a water medium—which may eventually serve as carriers for the previously mentioned substances.15–18 Materials used to create these gels have predominantly been of synthetic origin with predefined chemical and physical properties.19–22 Others have studied biological materials, suspensions that comprise molecules such as collagen I,23–25 laminin,26–28 dextran,29 chitosan,30,31 and hyaluronic acid.32,33 Although these hydrogels tend to induce healing in tissues and are clearly more biocompatible than their synthetic alternatives, they lack components of the ECM that are potentially critical for cellular migration, proliferation, and adhesion.34 Logically, a number of hydrogels have been developed from the very tissues that they aim to treat in an attempt to provide the native cues for cell-matrix interactions and injury response.35–41 These substances, solutions of various ECMs, have been shown to possess favorable cues for cellular infiltration, migration, and proliferation. A native tissue–derived microenvironment such as this could serve as the foundation on which healing may occur. Our previous research has described the manufacture of a hydrogel derived from human cadaveric tendon. Studies using this soluble ECM solution have demonstrated this hydrogel's potential to be a seeded with rat adipoderived stem cells (ASCs) while remaining biocompatible in vivo.42 Furthermore, our preliminary studies have revealed considerable efficacy in restoring the natural strength of tendon after injury in a rat model.43 Not only does this material retain its bioactivity, acting as a scaffold for cellular growth, ECM deposition, and remodeling, but also garners no immune response (a trait attributable to complete decellularization before processing). Tendon hydrogel seeded with fibroblasts or multipotent stem cells could allow for targeted regeneration at the site of injury and guide the restructuring of healing tissue in that specific region. An animal source of tendon hydrogel has not yet been described, but would prove beneficial due to the cost, availability, and ethical concerns associated with the harvest and use of human cadaveric tendon. This study evaluates the synthesis, physical characteristics, and biocompatibility of porcine tendon hydrogel as an alternative to human tendon hydrogel. The long-term aim of this work was to create an inexpensive, off-the-shelf injectable scaffold that provides a means for organized ECM synthesis and proper healing at the molecular level of a tendon injury.
METHODS Tissue Decellularization and Material Processing For the manufacture of human tendon hydrogel, flexor tendons were harvested from fresh cadaveric forearms at our institution. These
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tendons were meticulously debrided of remaining synovial material and muscle tissue. Flexor digitorum superficialis tendons were transected 2 cm proximal to the chiasma, while flexor digitorum profundus and flexor pollicis longus tendons were transected 1 cm proximal to the osteotendinous insertion. Harvested tendons were subsequently cut into 1-cm segments and treated with 0.1% ethylenediaminetetraacetic acid (EDTA) for 4 hours and then washed in 0.1% sodium dodecyl sulfate (SDS) in 0.1% EDTA for 24 hours at ambient temperature with constant agitation. Tendon was then rinsed in 1 PBS for 4 hours and stored at −80°C. For the manufacture of porcine tendon hydrogel, tendon was harvested from the dorsal and volar regions of fresh pig hindlimbs. In the same manner as its human counterpart, porcine tendon was meticulously debrided from remaining synovial material and muscle tissue. Harvested tendon was cut into 1-cm segments and treated with 0.2% EDTA for 4 hours followed by 0.2% SDS in 0.2% EDTA for 24 hours at ambient temperature with constant agitation. Tendon was then rinsed in 1 PBS for 4 hours and stored at −80°C. The frozen, decellularized tendons derived from human and porcine sources were lyophilized for 48 hours (Labconco, Kansas City, Mo), and milled into a fine powder using a Wiley Mini Mill, filter size 80 (Thomas Scientific, Swedesboro, NJ). The tendon powders were stored at 4°C until use.
Confirmation of Decellularization To assess for adequate decellularization, cell visualization via routine hematoxylin-eosin staining and SYTO Green 11 fluorescent staining was performed. In addition, DNeasy assay was used to quantify the amount of nucleic acid present in decellularized samples. First DNA was extracted from decellularized freeze-dried tendon. Using the absorbance peak at 260 nm as a proxy, the concentration of dsDNA present in the extract was quantified using an ultraviolet spectrophotometer (Biophotometer 22331; Eppendorf, Hauppauge, NY). The concentration of dsDNA present within samples was then extrapolated using a known extinction coefficient for the molecule.
ECM Gel Formation On the basis of previous optimization experiments, human tendon powder was enzymatically digested by the addition of 1 mg/mL pepsin in a 0.02 M HCl solution to obtain a final concentration of 20 mg/mL or 2%. The material was digested for 24 hours at ambient temperature with constant stirring to ensure homogeneity. Porcine tendon powder was similarly digested by the addition of 0.1 mg/mL pepsin in a 0.01 M HCl solution. Final concentrations of the material were 10, 15, and 20 mg/mL or 1%, 1.5%, and 2%, respectively. The material was digested for up to 24 hours at ambient temperature with constant stirring. Microscopic observation of the ECM
solution demonstrated that optimal digestion of milled porcine tendon powder occurred at 1 hour of stirring. Before use, human and porcine gel solutions were cooled on ice. Pepsin was denatured by the addition of 1 M NaOH until the pH of the mixture reached 8.5. The pH was subsequently adjusted to 7.4 by the addition of 1 M HCl. The salinity of the solution was also adjusted to physiologic levels by the addition of 10 PBS in a 1:10 fashion (1 mL 10 PBS per 10 mL gel).
Storage of Gel Human and porcine tendon hydrogels were stored at 4°C after proper digestion until use. Gels were left acidified at pH 2 until the time of the experiment. At that point, solutions were alkalized to denature pepsin and then brought to physiologic pH as described previously.
Mass Spectrometry Mass spectrometry (LTQ Orbitrap Velos; Thermo Scientific, Vancouver, Canada) was performed on milled decellularized porcine tendon before pepsin digestion. Analytic samples were prepared using a filter-aided sample preparation44 protocol. A small amount of the tendon powder was solubilized in SDS, DTT and Tris-HCl, and digested overnight by trypsin. The resulting peptide fragments were then loaded onto a self-packed fused silica C18 analytical column with a Bruker Michrom Advance interface (Auburn, Calif ). A flow rate of 600 nL/min and a spray voltage of 1.7 kV were used. The mass spectrometer was set in data-dependent acquisition mode and the 12 most intense precursor ions were detected. The data was converted to mzXML format and searched against the human uniprot-sprot database using Sequest on a Sorcerer platform. Scaffold 3 (Proteome Software, Portland, Ore) was used to analyze the data.
Rheology: Storage Modulus, Temperature Characteristics Rheological studies were performed with a TA Instruments ARG2 Rheometer. Gel was dispensed (500 μL) onto 2 parallel steel plates, one of which a probe with a 40-mm diameter. The plates were brought to a gap height of 100 μm, which allowed the gel to completely spread to the edge of the probe. Excess gel was gently wiped away. With a barrier for containment, silicone oil was used to prevent evaporation of the sample. The storage modulus (G′) and loss modulus (G″) were measured for range of frequencies (0.1–10 rad/s) at 25°C and 37°C (frequency sweep). The samples were then brought from 25°C to 37°C at a rate of 2°C per minute with a frequency 1 rad/s (temperature sweep). The temperature was then held at 37°C for 30 minutes and the storage and loss moduli were measured (time sweep).
FIGURE 1. Hematoxylin-eosin staining of native (A) and treated (B) porcine tendon (20). In contrast to the native tendon, no cells are observed between fibrous connective tissue. 48
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FIGURE 2. SYTO Green 11 fluorescent staining of native (A) and treated (B) porcine tendon (20). Decellularization is confirmed by the absence of fluorescent nuclei in the treated specimen.
Live/Dead Assay of Reseeded ASCs Human ASCs were gently mixed with hydrogels before incubation at 37°C. A live/dead assay (Promega, Madison, Wis) was performed on human and porcine hydrogels 7 days after seeding to assess the ratio of viable to nonviable cells. Cell-seeded hydrogels were gently mixed with the staining agents and incubated per standard protocol.
for 30 minutes. The contents of individual wells were transferred to microcentrifuge tubes and spun at 5000g for 2 minutes to pellet hydrogel and cells. The supernatant was then collected and plated for reading. Proliferation was represented by absorbance values (optical density) of the supernatant at 490 nm using an Epoch spectrophotometer (BioTek, Winooski, Vt).
RESULTS Cell Proliferation of Reseeded ASCs Cell proliferation in human and porcine gels was evaluated by the MTS colorimetric assay. Commercially derived human ASCs (up to passage 4, Poietics PT-5006 cryopreserved adipoderived stem cells, Lonza, Walkersville, MD) were cultured in fetal bovine serum (Gibco, Life Technologies, Grand Island, NY) supplemented ADSC-BM medium (Lonza). Adipoderived stem cells were grown until 90% confluence at 37°C in humidified tissue culture chamber with 5% CO2 content. Hydrogel was then seeded with ASCs. Proliferation was measured at 3 and 5 days. Tetrazolium dye (Cell Titer 96 AQueous Nonradioactive Cell Proliferation Assay; Promega) was directly added to wells in a 1:10 fashion, gently mixed, and allowed to incubate at 37°C
Manufacture of Human and Porcine Gel Human tendon was successfully decellularized based on previously described methods. However, this particular decellularization protocol was unable to remove cells from porcine tendon. Increasing concentrations of wash solutions were attempted until decellularization was achieved with 0.2% EDTA followed by 0.2% EDTA in 0.2% SDS. Routine hematoxylin-eosin staining (Fig. 1A) and SYTO Green 11 fluorescent staining (Fig. 2B) confirmed decellularization. DNeasy assay demonstrated a concentration of 132 ng dsDNA per milligram decellularized tendon, a value similar to that obtained from decellularized human tendon.45
FIGURE 3. Digestion optimization of human and porcine tendon hydrogels. © 2014 Wolters Kluwer Health, Inc. All rights reserved. Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.
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Collagen 6A3 was the most abundant single protein in the sample (17.1% of the total content) followed by collagen 6A2 (9.2%). A number of other ECM proteins were also found in the sample.
Rheology The rheological properties of 2% human and 1% porcine hydrogels were determined through triplicates of the storage (G′) and loss moduli (G″). Representative samples are shown graphically (Fig. 5). Frequency sweeps were performed to demonstrate the elastic and viscous tendencies of the samples at a range of angular frequencies as shown in Figures 5A and B. At 25°C, G′ at 1 rad/s was found to be 177.9 and 137.9, whereas G″ was 27.6 and 34.1 for human and porcine
FIGURE 4. Protein composition of porcine tendon powder pre-digestion and post-digestion. Collagen IV and Collagen I were the predominant proteins found both samples. Other peptides included tenascin, vimentin, fibromodulin, decorin, and a number other extracellular proteins.
ECM Gel Formation—Digestion Time and Concentration Human hydrogel at a concentration of 20 mg/mL (2%) was successfully prepared based on methods and optimization experiments outlined in our prior study. Porcine hydrogel concentrations ranging from 10 (1%) to 20 (2%) mg/mL were prepared. Because of the handling properties, cellular proliferation profile, and structural integrity of 1% porcine gel, this specific ECM concentration was chosen for rheological, biocompatibility, and proliferation studies. Human hydrogel was digested for 24 hours with 1 mg/mL pepsin. Porcine hydrogel was digested for only 1 hour with 0.1 mg/mL pepsin. Our previous work describes how to assess for proper digestion microscopically. Figure 3 details human and porcine gel samples that have undergone overoptimal, underoptimal, and optimal digestion.
Storage of Gel Extracellular matrix solutions gradually lost their ability to form gels over ensuing weeks unless the samples were stored at pH 2.2. At pH 2.2, liquefied ECM could be stored for prolonged periods, with unchanged gelation properties after neutralization to pH 7.4.
Mass Spectrometry Proteome analysis of porcine tendon powder was performed after filter-aided sample preparation and mass spectroscopy, which yielded 150 of the most abundant proteins in the sample. The 15 most abundant proteins are listed in Figure 4. Collagen represented the most predominant protein family, constituting 32.3% of the total sample. 50
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FIGURE 5. Frequency sweep of human and porcine tendon hydrogels; 25°C (A). Temperature sweep of human and porcine tendon hydrogels (B). Time sweep of human and porcine tendon hydrogels; 37°C (C). © 2014 Wolters Kluwer Health, Inc. All rights reserved.
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FIGURE 6. Morphology of human ASCs seeded within 2% human (A) and 1% porcine (B) tendon hydrogels. ASCs retain their spindle-like morphology in each ECM solution.
samples, respectively. At 37°C, G′ at 1 rad/s was found to be 236.0 and 113.2, whereas G″ was 22.5 and 12.4 for human and porcine samples, respectively. To determine the effect of temperature on G′ and G″, a temperature sweep was performed (Fig. 5C). As the temperature increased from 25°C to 37°C at a rate of 2°C per minute, the storage modulus for human tendon hydrogel increased from 180.6 to 208.3. Yet, the storage modulus for porcine tendon hydrogel did not share this trend, decreasing from 139.8 to 108.0. Once the temperature reached 37°C, the ECM solutions were monitored without further increases in temperature via a time sweep. In both human and porcine samples, the storage modulus (G′) of minimally increased while the loss modulus (G″) minimally decreased as a function of time (Fig. 5C).
In Vitro—Live/Dead Staining and Cell Morphology For human and porcine samples alike, spindle-shaped cells were observed (Fig. 6), displaying an even distribution throughout the gel
solution. Human ASCs were gently suspended within ECM solutions, and cell viability was measured 7 days after seeding. During this time course, the fraction of nonviable cells remained low as assessed visually by live/dead assay (Fig. 7).
Cellular Proliferation Human ASCs were seeded within human and porcine tendon hydrogels. At days 3 and 5, there was minimal difference between the proliferation of cells with human and porcine samples as determined by tetrazolium dye assay (Fig. 8).
DISCUSSION Porcine xenografts have widely been used in medicine during the last 40 years, owing largely to their availability and donor compatibility. For several decades, pig valves have been grafted into patients with valvular heart disease.46 To avoid rejection of live tissue, valves were treated with glutaraldehyde (a fixing agent) to allow for the graft
FIGURE 7. Live/dead assay. The ratio of viable to nonviable cells remained low for human ASCs within human and porcine tendon hydrogels. Viable cells are seen as green and nonviable cells are seen as red. © 2014 Wolters Kluwer Health, Inc. All rights reserved. Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.
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FIGURE 8. Human ASC proliferation in human and porcine hydrogel at days 3 and 5.
to be “immunologically inert.”47 Although glutaraldehyde stabilization was found to lead to calcifications,48 processing and decellularization still remain a necessity for the clinical translation of xenografts. Another porcine-derived tissue is dermal collagen, a substance that has been of great value in soft tissue reconstruction. Similarly, processing of these grafts comprises strict decellularization, removal of other antigenic material, and depilation—leaving behind a collagen framework.49 To achieve this result, dermis is generally agitated to allow for greater porosity, washed with SDS or other detergents, and enzymatically digestion.50–52 In the current study, porcine tendon proved slightly different from human tendon in a few ways; notably, it was more difficult to decellularize, a trait attributable to the density of the connective tissue found in pig tendon. Despite its original toughness, once in powder form, the porcine tendon required far less digestion (in regards to time and concentration of enzyme) than its human counterpart. The handling properties of the sample, an essential attribute if hydrogel is to be administered clinically, were also evaluated. Human hydrogel at a concentration of 2% can easily be pipetted and applied at milliliter and microliter volumes. Porcine hydrogel, on the other hand, proved more viscous at that same concentration. We found that the viscosity of 1% porcine hydrogel most resembled that of its 2% human counterpart. At this concentration, porcine hydrogel was easily manipulated and could similarly be pipetted at a range of different volumes. It has been previously noted that the presence of fibrinogen strands within the gel are correlated with its ability to gelate at 37°C. Overdigested samples demonstrated the complete absence of these fibers, whereas underdigested samples showed a heterogeneous mixture of solid tendon material. Rheological studies, however, indicate the porcine hydrogel does not become as solid and elastic as human hydrogel, melting slightly as the temperature increase. Despite this tendency, both specimens tend to increase their storage modulus over time at body temperature. Some of this variability between human and porcine gels may be attributed to the difference in concentration (2% human vs 1% porcine). Despite possessing a different modulus profile from its human counterpart, porcine hydrogel remains a viscous gel at a range of temperature. Because its viscosity remains relatively stable over a range of temperatures, injectable porcine hydrogel will likely hold its shape and conform to the space in which it was dispensed. Porcine tendon powder consisted largely of collagen and other ECM proteins. Collagen VI was found to be the predominant protein in the sample. This form of collagen is largely found in muscle, suggesting incomplete debridement of surrounding muscle tissue. However, very little actin and myosin were found within the sample, which would discourage this assumption. We hypothesize that the collagen found within the sample, in addition to the number of other 52
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ECM proteins, provide a microenvironment, which is suitable for cell growth and proliferation. Porcine tendon hydrogel provided a biocompatible scaffold for human ASCs to proliferate. No difference existed between cellular proliferation in human and porcine gels, providing evidence that porcine tendon hydrogel could be seeded with human cells that would persist at the site of injury. In conclusion, porcine tendon hydrogel shared many key characteristics of human hydrogel, most notably the ability to sustain the growth of human cells. This novel porcine hydrogel material has potential as an injectable material that would promote tendon regeneration and healing in tendinopathies and other tendon injuries.
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In Vitro Characteristics of Porcine Tendon Hydrogel for Tendon Regeneration Christopher S. Crowe, BS,*† Grace Chiou, MD,*† Rory McGoldrick, MBBS, MBA,*† Kenneth Hui, BA,*† Hung Pham, BS,*† Emily Hollenbeck, BS,*‡ and James Chang, MD*† Purpose: Previous work has characterized the development of a human tendon hydrogel capable of improving mechanical strength after tendon injury. Animal tendon hydrogel has not yet been described, but would prove beneficial due to the cost and ethical concerns associated with the use of human cadaveric tendon. This study details the manufacture and assesses the biocompatibility of porcine tendon hydrogel seeded with human adipoderived stem cells (ASCs). Materials and Methods: Porcine tendon was dissected from surrounding connective and muscle tissue and decellularized via 0.2% sodium dodecyl sulfate and 0.2% sodium dodecyl sulfate/ethylenediaminetetraacetic acid wash solutions before lyophilization. Tendon was milled and reconstituted by previously described methods. Decellularization was confirmed by hematoxylin-eosin staining, SYTO Green 11 nucleic acid dye, and DNeasy assay. The protein composition of milled tendon matrix before and after digestion was identified by mass spectrometry. Rheological properties were determined using an ARG2 rheometer. Biocompatibility was assessed by live/dead assay. The proliferation of human ASCs seeded in porcine and human hydrogel was measured by MTS assay. All experimental conditions were performed in triplicate. Results: Decellularization of porcine tendon was successful. Mass spectrometry showed that collagen composes one third of milled porcine tendon before and after pepsin digestion. Rheology demonstrated that porcine hydrogel maintains a fluid consistency over a range of temperatures, unlike human hydrogel, which tends to solidify. Live/dead staining revealed that human ASCs survive in hydrogel 7 days after seeding and retain spindle-like morphology. MTS assay at day 3 and day 5 showed that human ASC proliferation was marginally greater in human hydrogel. Conclusions: After reconstitution and digestion, porcine hydrogel was capable of supporting growth of human ASCs. The minimal difference in proliferative capacity suggests that porcine tendon hydrogel may be an effective and viable alternative to human hydrogel for the enhancement of tendon healing. Key Words: tissue engineering, porcine tendon, tendon repair, xenograft (Ann Plast Surg 2016;77: 47–53)
T
endon healing represents a complex process characterized by both intrinsic and extrinsic mechanisms.1 After injury, tenocytes are capable of proliferating and migrating to the site of repair. In addition, healing may also be driven by the ingrowth of other local cells and vascularization from adjacent tissue. The biology of tendon healing becomes clinically relevant when viewed as the basis of functional outcomes. With repetitive stress, such as the case with tendinopathy, the balance between extracellular matrix (ECM) synthesis and degradation can be tipped.2,3 Additionally, disorganized synthesis of collagen fibrils Received July 28, 2014, and accepted for publication, after revision, September 1, 2014. From the *Division of Plastic and Reconstructive Surgery, Stanford University Medical Center, Stanford; †Section of Plastic Surgery, VA Palo Alto Health Care System, Palo Alto; and ‡Department of Chemical Engineering, Stanford University, Stanford, CA. Conflicts of interest and sources of funding: The laboratory is funded by several Federal grants including the VA RR&D Merit Review program. Reprints: James Chang, MD, Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University Medical Center, Stanford, CA. E-mail: [email protected]. Copyright © 2014 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0148-7043/16/7701–0047 DOI: 10.1097/SAP.0000000000000361
may be noted microscopically,4,5 translating into diminished tensile strength of the tissue. Understanding how tendon healing proceeds, and more importantly how it fails, provides considerable insight into the challenges that new interventions must mitigate. Because tendon repair follows a slow course that is complicated by many factors, a variety of methods to stimulate and aid healing have been attempted. These include injections of whole blood,6 plateletrich plasma,7–9 multipotent stem cells,10–12 and combinations of growth factors and hormones.6,13,14 Some attention has been paid to injectable hydrogels—particles dispersed within a water medium—which may eventually serve as carriers for the previously mentioned substances.15–18 Materials used to create these gels have predominantly been of synthetic origin with predefined chemical and physical properties.19–22 Others have studied biological materials, suspensions that comprise molecules such as collagen I,23–25 laminin,26–28 dextran,29 chitosan,30,31 and hyaluronic acid.32,33 Although these hydrogels tend to induce healing in tissues and are clearly more biocompatible than their synthetic alternatives, they lack components of the ECM that are potentially critical for cellular migration, proliferation, and adhesion.34 Logically, a number of hydrogels have been developed from the very tissues that they aim to treat in an attempt to provide the native cues for cell-matrix interactions and injury response.35–41 These substances, solutions of various ECMs, have been shown to possess favorable cues for cellular infiltration, migration, and proliferation. A native tissue–derived microenvironment such as this could serve as the foundation on which healing may occur. Our previous research has described the manufacture of a hydrogel derived from human cadaveric tendon. Studies using this soluble ECM solution have demonstrated this hydrogel's potential to be a seeded with rat adipoderived stem cells (ASCs) while remaining biocompatible in vivo.42 Furthermore, our preliminary studies have revealed considerable efficacy in restoring the natural strength of tendon after injury in a rat model.43 Not only does this material retain its bioactivity, acting as a scaffold for cellular growth, ECM deposition, and remodeling, but also garners no immune response (a trait attributable to complete decellularization before processing). Tendon hydrogel seeded with fibroblasts or multipotent stem cells could allow for targeted regeneration at the site of injury and guide the restructuring of healing tissue in that specific region. An animal source of tendon hydrogel has not yet been described, but would prove beneficial due to the cost, availability, and ethical concerns associated with the harvest and use of human cadaveric tendon. This study evaluates the synthesis, physical characteristics, and biocompatibility of porcine tendon hydrogel as an alternative to human tendon hydrogel. The long-term aim of this work was to create an inexpensive, off-the-shelf injectable scaffold that provides a means for organized ECM synthesis and proper healing at the molecular level of a tendon injury.
METHODS Tissue Decellularization and Material Processing For the manufacture of human tendon hydrogel, flexor tendons were harvested from fresh cadaveric forearms at our institution. These
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tendons were meticulously debrided of remaining synovial material and muscle tissue. Flexor digitorum superficialis tendons were transected 2 cm proximal to the chiasma, while flexor digitorum profundus and flexor pollicis longus tendons were transected 1 cm proximal to the osteotendinous insertion. Harvested tendons were subsequently cut into 1-cm segments and treated with 0.1% ethylenediaminetetraacetic acid (EDTA) for 4 hours and then washed in 0.1% sodium dodecyl sulfate (SDS) in 0.1% EDTA for 24 hours at ambient temperature with constant agitation. Tendon was then rinsed in 1 PBS for 4 hours and stored at −80°C. For the manufacture of porcine tendon hydrogel, tendon was harvested from the dorsal and volar regions of fresh pig hindlimbs. In the same manner as its human counterpart, porcine tendon was meticulously debrided from remaining synovial material and muscle tissue. Harvested tendon was cut into 1-cm segments and treated with 0.2% EDTA for 4 hours followed by 0.2% SDS in 0.2% EDTA for 24 hours at ambient temperature with constant agitation. Tendon was then rinsed in 1 PBS for 4 hours and stored at −80°C. The frozen, decellularized tendons derived from human and porcine sources were lyophilized for 48 hours (Labconco, Kansas City, Mo), and milled into a fine powder using a Wiley Mini Mill, filter size 80 (Thomas Scientific, Swedesboro, NJ). The tendon powders were stored at 4°C until use.
Confirmation of Decellularization To assess for adequate decellularization, cell visualization via routine hematoxylin-eosin staining and SYTO Green 11 fluorescent staining was performed. In addition, DNeasy assay was used to quantify the amount of nucleic acid present in decellularized samples. First DNA was extracted from decellularized freeze-dried tendon. Using the absorbance peak at 260 nm as a proxy, the concentration of dsDNA present in the extract was quantified using an ultraviolet spectrophotometer (Biophotometer 22331; Eppendorf, Hauppauge, NY). The concentration of dsDNA present within samples was then extrapolated using a known extinction coefficient for the molecule.
ECM Gel Formation On the basis of previous optimization experiments, human tendon powder was enzymatically digested by the addition of 1 mg/mL pepsin in a 0.02 M HCl solution to obtain a final concentration of 20 mg/mL or 2%. The material was digested for 24 hours at ambient temperature with constant stirring to ensure homogeneity. Porcine tendon powder was similarly digested by the addition of 0.1 mg/mL pepsin in a 0.01 M HCl solution. Final concentrations of the material were 10, 15, and 20 mg/mL or 1%, 1.5%, and 2%, respectively. The material was digested for up to 24 hours at ambient temperature with constant stirring. Microscopic observation of the ECM
solution demonstrated that optimal digestion of milled porcine tendon powder occurred at 1 hour of stirring. Before use, human and porcine gel solutions were cooled on ice. Pepsin was denatured by the addition of 1 M NaOH until the pH of the mixture reached 8.5. The pH was subsequently adjusted to 7.4 by the addition of 1 M HCl. The salinity of the solution was also adjusted to physiologic levels by the addition of 10 PBS in a 1:10 fashion (1 mL 10 PBS per 10 mL gel).
Storage of Gel Human and porcine tendon hydrogels were stored at 4°C after proper digestion until use. Gels were left acidified at pH 2 until the time of the experiment. At that point, solutions were alkalized to denature pepsin and then brought to physiologic pH as described previously.
Mass Spectrometry Mass spectrometry (LTQ Orbitrap Velos; Thermo Scientific, Vancouver, Canada) was performed on milled decellularized porcine tendon before pepsin digestion. Analytic samples were prepared using a filter-aided sample preparation44 protocol. A small amount of the tendon powder was solubilized in SDS, DTT and Tris-HCl, and digested overnight by trypsin. The resulting peptide fragments were then loaded onto a self-packed fused silica C18 analytical column with a Bruker Michrom Advance interface (Auburn, Calif ). A flow rate of 600 nL/min and a spray voltage of 1.7 kV were used. The mass spectrometer was set in data-dependent acquisition mode and the 12 most intense precursor ions were detected. The data was converted to mzXML format and searched against the human uniprot-sprot database using Sequest on a Sorcerer platform. Scaffold 3 (Proteome Software, Portland, Ore) was used to analyze the data.
Rheology: Storage Modulus, Temperature Characteristics Rheological studies were performed with a TA Instruments ARG2 Rheometer. Gel was dispensed (500 μL) onto 2 parallel steel plates, one of which a probe with a 40-mm diameter. The plates were brought to a gap height of 100 μm, which allowed the gel to completely spread to the edge of the probe. Excess gel was gently wiped away. With a barrier for containment, silicone oil was used to prevent evaporation of the sample. The storage modulus (G′) and loss modulus (G″) were measured for range of frequencies (0.1–10 rad/s) at 25°C and 37°C (frequency sweep). The samples were then brought from 25°C to 37°C at a rate of 2°C per minute with a frequency 1 rad/s (temperature sweep). The temperature was then held at 37°C for 30 minutes and the storage and loss moduli were measured (time sweep).
FIGURE 1. Hematoxylin-eosin staining of native (A) and treated (B) porcine tendon (20). In contrast to the native tendon, no cells are observed between fibrous connective tissue. 48
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FIGURE 2. SYTO Green 11 fluorescent staining of native (A) and treated (B) porcine tendon (20). Decellularization is confirmed by the absence of fluorescent nuclei in the treated specimen.
Live/Dead Assay of Reseeded ASCs Human ASCs were gently mixed with hydrogels before incubation at 37°C. A live/dead assay (Promega, Madison, Wis) was performed on human and porcine hydrogels 7 days after seeding to assess the ratio of viable to nonviable cells. Cell-seeded hydrogels were gently mixed with the staining agents and incubated per standard protocol.
for 30 minutes. The contents of individual wells were transferred to microcentrifuge tubes and spun at 5000g for 2 minutes to pellet hydrogel and cells. The supernatant was then collected and plated for reading. Proliferation was represented by absorbance values (optical density) of the supernatant at 490 nm using an Epoch spectrophotometer (BioTek, Winooski, Vt).
RESULTS Cell Proliferation of Reseeded ASCs Cell proliferation in human and porcine gels was evaluated by the MTS colorimetric assay. Commercially derived human ASCs (up to passage 4, Poietics PT-5006 cryopreserved adipoderived stem cells, Lonza, Walkersville, MD) were cultured in fetal bovine serum (Gibco, Life Technologies, Grand Island, NY) supplemented ADSC-BM medium (Lonza). Adipoderived stem cells were grown until 90% confluence at 37°C in humidified tissue culture chamber with 5% CO2 content. Hydrogel was then seeded with ASCs. Proliferation was measured at 3 and 5 days. Tetrazolium dye (Cell Titer 96 AQueous Nonradioactive Cell Proliferation Assay; Promega) was directly added to wells in a 1:10 fashion, gently mixed, and allowed to incubate at 37°C
Manufacture of Human and Porcine Gel Human tendon was successfully decellularized based on previously described methods. However, this particular decellularization protocol was unable to remove cells from porcine tendon. Increasing concentrations of wash solutions were attempted until decellularization was achieved with 0.2% EDTA followed by 0.2% EDTA in 0.2% SDS. Routine hematoxylin-eosin staining (Fig. 1A) and SYTO Green 11 fluorescent staining (Fig. 2B) confirmed decellularization. DNeasy assay demonstrated a concentration of 132 ng dsDNA per milligram decellularized tendon, a value similar to that obtained from decellularized human tendon.45
FIGURE 3. Digestion optimization of human and porcine tendon hydrogels. © 2014 Wolters Kluwer Health, Inc. All rights reserved. Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.
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Collagen 6A3 was the most abundant single protein in the sample (17.1% of the total content) followed by collagen 6A2 (9.2%). A number of other ECM proteins were also found in the sample.
Rheology The rheological properties of 2% human and 1% porcine hydrogels were determined through triplicates of the storage (G′) and loss moduli (G″). Representative samples are shown graphically (Fig. 5). Frequency sweeps were performed to demonstrate the elastic and viscous tendencies of the samples at a range of angular frequencies as shown in Figures 5A and B. At 25°C, G′ at 1 rad/s was found to be 177.9 and 137.9, whereas G″ was 27.6 and 34.1 for human and porcine
FIGURE 4. Protein composition of porcine tendon powder pre-digestion and post-digestion. Collagen IV and Collagen I were the predominant proteins found both samples. Other peptides included tenascin, vimentin, fibromodulin, decorin, and a number other extracellular proteins.
ECM Gel Formation—Digestion Time and Concentration Human hydrogel at a concentration of 20 mg/mL (2%) was successfully prepared based on methods and optimization experiments outlined in our prior study. Porcine hydrogel concentrations ranging from 10 (1%) to 20 (2%) mg/mL were prepared. Because of the handling properties, cellular proliferation profile, and structural integrity of 1% porcine gel, this specific ECM concentration was chosen for rheological, biocompatibility, and proliferation studies. Human hydrogel was digested for 24 hours with 1 mg/mL pepsin. Porcine hydrogel was digested for only 1 hour with 0.1 mg/mL pepsin. Our previous work describes how to assess for proper digestion microscopically. Figure 3 details human and porcine gel samples that have undergone overoptimal, underoptimal, and optimal digestion.
Storage of Gel Extracellular matrix solutions gradually lost their ability to form gels over ensuing weeks unless the samples were stored at pH 2.2. At pH 2.2, liquefied ECM could be stored for prolonged periods, with unchanged gelation properties after neutralization to pH 7.4.
Mass Spectrometry Proteome analysis of porcine tendon powder was performed after filter-aided sample preparation and mass spectroscopy, which yielded 150 of the most abundant proteins in the sample. The 15 most abundant proteins are listed in Figure 4. Collagen represented the most predominant protein family, constituting 32.3% of the total sample. 50
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FIGURE 5. Frequency sweep of human and porcine tendon hydrogels; 25°C (A). Temperature sweep of human and porcine tendon hydrogels (B). Time sweep of human and porcine tendon hydrogels; 37°C (C). © 2014 Wolters Kluwer Health, Inc. All rights reserved.
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FIGURE 6. Morphology of human ASCs seeded within 2% human (A) and 1% porcine (B) tendon hydrogels. ASCs retain their spindle-like morphology in each ECM solution.
samples, respectively. At 37°C, G′ at 1 rad/s was found to be 236.0 and 113.2, whereas G″ was 22.5 and 12.4 for human and porcine samples, respectively. To determine the effect of temperature on G′ and G″, a temperature sweep was performed (Fig. 5C). As the temperature increased from 25°C to 37°C at a rate of 2°C per minute, the storage modulus for human tendon hydrogel increased from 180.6 to 208.3. Yet, the storage modulus for porcine tendon hydrogel did not share this trend, decreasing from 139.8 to 108.0. Once the temperature reached 37°C, the ECM solutions were monitored without further increases in temperature via a time sweep. In both human and porcine samples, the storage modulus (G′) of minimally increased while the loss modulus (G″) minimally decreased as a function of time (Fig. 5C).
In Vitro—Live/Dead Staining and Cell Morphology For human and porcine samples alike, spindle-shaped cells were observed (Fig. 6), displaying an even distribution throughout the gel
solution. Human ASCs were gently suspended within ECM solutions, and cell viability was measured 7 days after seeding. During this time course, the fraction of nonviable cells remained low as assessed visually by live/dead assay (Fig. 7).
Cellular Proliferation Human ASCs were seeded within human and porcine tendon hydrogels. At days 3 and 5, there was minimal difference between the proliferation of cells with human and porcine samples as determined by tetrazolium dye assay (Fig. 8).
DISCUSSION Porcine xenografts have widely been used in medicine during the last 40 years, owing largely to their availability and donor compatibility. For several decades, pig valves have been grafted into patients with valvular heart disease.46 To avoid rejection of live tissue, valves were treated with glutaraldehyde (a fixing agent) to allow for the graft
FIGURE 7. Live/dead assay. The ratio of viable to nonviable cells remained low for human ASCs within human and porcine tendon hydrogels. Viable cells are seen as green and nonviable cells are seen as red. © 2014 Wolters Kluwer Health, Inc. All rights reserved. Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.
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FIGURE 8. Human ASC proliferation in human and porcine hydrogel at days 3 and 5.
to be “immunologically inert.”47 Although glutaraldehyde stabilization was found to lead to calcifications,48 processing and decellularization still remain a necessity for the clinical translation of xenografts. Another porcine-derived tissue is dermal collagen, a substance that has been of great value in soft tissue reconstruction. Similarly, processing of these grafts comprises strict decellularization, removal of other antigenic material, and depilation—leaving behind a collagen framework.49 To achieve this result, dermis is generally agitated to allow for greater porosity, washed with SDS or other detergents, and enzymatically digestion.50–52 In the current study, porcine tendon proved slightly different from human tendon in a few ways; notably, it was more difficult to decellularize, a trait attributable to the density of the connective tissue found in pig tendon. Despite its original toughness, once in powder form, the porcine tendon required far less digestion (in regards to time and concentration of enzyme) than its human counterpart. The handling properties of the sample, an essential attribute if hydrogel is to be administered clinically, were also evaluated. Human hydrogel at a concentration of 2% can easily be pipetted and applied at milliliter and microliter volumes. Porcine hydrogel, on the other hand, proved more viscous at that same concentration. We found that the viscosity of 1% porcine hydrogel most resembled that of its 2% human counterpart. At this concentration, porcine hydrogel was easily manipulated and could similarly be pipetted at a range of different volumes. It has been previously noted that the presence of fibrinogen strands within the gel are correlated with its ability to gelate at 37°C. Overdigested samples demonstrated the complete absence of these fibers, whereas underdigested samples showed a heterogeneous mixture of solid tendon material. Rheological studies, however, indicate the porcine hydrogel does not become as solid and elastic as human hydrogel, melting slightly as the temperature increase. Despite this tendency, both specimens tend to increase their storage modulus over time at body temperature. Some of this variability between human and porcine gels may be attributed to the difference in concentration (2% human vs 1% porcine). Despite possessing a different modulus profile from its human counterpart, porcine hydrogel remains a viscous gel at a range of temperature. Because its viscosity remains relatively stable over a range of temperatures, injectable porcine hydrogel will likely hold its shape and conform to the space in which it was dispensed. Porcine tendon powder consisted largely of collagen and other ECM proteins. Collagen VI was found to be the predominant protein in the sample. This form of collagen is largely found in muscle, suggesting incomplete debridement of surrounding muscle tissue. However, very little actin and myosin were found within the sample, which would discourage this assumption. We hypothesize that the collagen found within the sample, in addition to the number of other 52
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ECM proteins, provide a microenvironment, which is suitable for cell growth and proliferation. Porcine tendon hydrogel provided a biocompatible scaffold for human ASCs to proliferate. No difference existed between cellular proliferation in human and porcine gels, providing evidence that porcine tendon hydrogel could be seeded with human cells that would persist at the site of injury. In conclusion, porcine tendon hydrogel shared many key characteristics of human hydrogel, most notably the ability to sustain the growth of human cells. This novel porcine hydrogel material has potential as an injectable material that would promote tendon regeneration and healing in tendinopathies and other tendon injuries.
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