Page 1 of 46
Tissue Engineering Part A Design and Characterization of an Injectable Tendon Hydrogel: A Scaffold for Guided Tissue Regeneration in the Musculoskeletal System (doi: 10.1089/ten.TEA.2013.0207) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
1
Design and Characterization of an Injectable Tendon Hydrogel: A Novel Scaffold for Guided Tissue Regeneration in the Musculoskeletal System
Simon Farnebo1, Colin Y Woon1, Taliah Schmitt1, Lydia-Marie Joubert 2, Max Kim1, Hung Pham1, James Chang1
1
Division of Plastic and Reconstructive Surgery, Stanford University Medical Center
and Section of Plastic Surgery, VA Palo Alto Health Care System 2
Cell Sciences Imaging Facility, Stanford University Medical School, Stanford.
Keywords: Tendon, ECM (extracellular matrix), hydrogel, tissue engineering, scaffold
Corresponding author: James Chang MD Division of Plastic and Reconstructive Surgery Stanford University Medical Center 770 Welch Road, Suite 400 Stanford, CA 94304 [email protected]
Page 2 of 46
Tissue Engineering Part A Design and Characterization of an Injectable Tendon Hydrogel: A Scaffold for Guided Tissue Regeneration in the Musculoskeletal System (doi: 10.1089/ten.TEA.2013.0207) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
2 Abstract: A biocompatible hydrogel consisting of extracellular matrix (ECM) from human tendons is described as a potential scaffold for guided tissue regeneration and tissue engineering purposes. Lyophilized decellularized tendons were milled and enzymatically digested to form an ECM solution. The ECM solution properties are assessed by proteome analysis with mass spectrometry, and the material’s rheological properties are determined both as a function of frequency, temperature and time. In vivo application of the gel in a rat model is assessed for remodeling and host cell repopulation. Histology for macrophage invasion, fibroblast repopulation and nanoscale properties of the gel is assessed. Gel interaction with multipotent adipoderived stem cells (ASCs) is also addressed in vitro to assess possible cytotoxicity and its ability to act as a delivery vehicle for cells. Proteome analysis of the ECM-solution and gel mass spectroscopy identified the most abundant 150 proteins, of which two isoforms of Collagen I represented more than 55% of the sample. Rheology showed that storage (G’) and loss (G”) of the ECM solution were stable at room temperature but displayed sigmoidal increases after approximately 15 minutes at 37°C, matching macroscopic observations of its thermo responsiveness. G’ and G” of the gel at 1 rad/s were 213.1±19.9 Pa and 27.1±2.4 Pa respectively. Electron microscopy revealed fiber alignment and good structural porosity in the gel, as well as invasion of cells in vivo. Histology also showed early CD 68+ macrophage invasion throughout the gel, followed by increasing numbers of fibroblast cells. ASCs mixed with the gel in vitro proliferated, indicating good biocompatibility. This ECM solution can be delivered percutaneously into a zone of tendon injury. After injection, the thermoresponsive behavior of the ECM solution allows it to polymerize and form a porous gel at body temperature. A supportive nanostructure of collagen fibers is
Page 3 of 46
Tissue Engineering Part A Design and Characterization of an Injectable Tendon Hydrogel: A Scaffold for Guided Tissue Regeneration in the Musculoskeletal System (doi: 10.1089/ten.TEA.2013.0207) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
3 established that conforms to the three dimensional space of the defect. This hydrogel holds the distinctive composition specific for tendon ECM, where tissue specific cues facilitate host cell infiltration and remodeling. The results presented indicate that injectable ECM materials from tendon may offer a promising alternative in the treatment of tendinopathies and acute tendon injuries.
Page 4 of 46
Tissue Engineering Part A Design and Characterization of an Injectable Tendon Hydrogel: A Scaffold for Guided Tissue Regeneration in the Musculoskeletal System (doi: 10.1089/ten.TEA.2013.0207) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
4 Introduction: Ligament and tendon healing are important aspects of orthopedic surgery, hand surgery and sports medicine. In tendinopathy, tendon overuse leads to significant histological and biochemical changes that alter biomechanical and material properties 1, causing accumulative microscopic tears that ultimately result in full-thickness tears 2. These injuries are most commonly seen in the rotator cuff, Achilles tendon, quadriceps tendon, and patella tendon. In addition, medial and lateral epicondylitis of the elbow, and other chronic enthesopathies (disorders of the tendon-bone attachment), are believed to involve similar mechanisms of injury. Microstructural changes seen include micro-tears, thinning and disorganization of collagen fibers 3, 4, neovascularization of the tendon matrix 5, and morphological changes to tenocytes 6. Methods attempted by various authors to augment and stimulate healing 7 include injections of platelet rich plasma (PRP) 8 9 10, whole blood 11, or growth hormones 11-13; and the addition of multipotent stem cells. 14-16 Ligament and tendon healing may be accelerated by direct delivery of biocompatible scaffolds to the zone of injury that would facilitate in situ regeneration by means of threedimensional (3d) guided tissue regeneration 17. The 3d-scaffold would support the physical properties of the tissue and facilitate migration of surrounding cells into the scaffold microenvironment. These cells would subsequently proliferate and replace the scaffold with regenerated tissue. The application of a biodegradable scaffold is attractive as it may act both as a structural matrix and as a three-dimensional carrier of cells or growth hormones. 18 19 20 21
Most of the investigated injectable hydrogels have been synthetic polymers 22-24 with defined structural, chemical, and mechanical properties designed for a desired application, or composed of individual components of the ECM. Gels composed of type I collagen 25-27, hyaluronic acid 28, 29, or laminin 30-32 display desirable biocompatibility and strength.
Page 5 of 46
Tissue Engineering Part A Design and Characterization of an Injectable Tendon Hydrogel: A Scaffold for Guided Tissue Regeneration in the Musculoskeletal System (doi: 10.1089/ten.TEA.2013.0207) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
5 However, when taken out of their ECM context, they do not appropriately match the injured microenvironment and therefore lose some of the essential cues that would be needed for cellular repopulation. There have been efforts to develop injectable hydrogels that are derived from the tissue being treated or replaced. These gels would be a better match as they would possess an ECM composition matched to the native tissue, as well as the chemical and biological cues inherent to these matrix components 33 34 35 36 37 38 39 . A hydrogel based on decellularized materials will thus have a distinct composition that closely matches the tissue of origin, 40 34 allowing for the development of tissue specific scaffolds for appropriate cell–matrix interactions. Specific to tendon repair and regeneration, collagen fibril segments (CFS) are tendon ECM elements used in fetal tendon development 41, 42. Numerous studies suggest that intrinsic tendon repair uses recycled CFS as an immediate source in reconnecting severed fibrils during tendon healing 42-45, and that endogenous tendon fibroblasts will bridge the gap between the severed tendon ends with the deposition of new connective tissue matrix as part of an intrinsic tendon repair. It is proposed that released CFS from recycled collagen fibers could generate a pool of CFS that supply a portion of the building blocks utilized at the healing site 42, 44. These studies do not directly address the issue of ECM element placement and delivery. Directed delivery of ECM elements using needle injection, or implantation during surgery, or using delivery vehicles such as hydrogel-impregnated collagen sponges or hydrogel-coated sutures may address the two-fold objective of providing native materials, and placing ECM components, such as collagen fibril segments (CFS) from recycled collagen fibers directly at the repair site, where active ECM synthesis is taking place. Although type I collagen is the major ECM element involved, it is possible that other
Page 6 of 46
Tissue Engineering Part A Design and Characterization of an Injectable Tendon Hydrogel: A Scaffold for Guided Tissue Regeneration in the Musculoskeletal System (doi: 10.1089/ten.TEA.2013.0207) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
6 ECM components are involved in repair and an ECM-rich solution composed of tendon digestate may augment tendon healing to an extent greater than purified CFS alone. An ECM solution with appropriate viscosity can be delivered in a minimally invasive fashion by injection, and then conform to the three-dimensional space following deposition. It has been shown previously that the thermoresponsive behavior of different ECM solutions allows it to polymerize and form a gel at body temperature, creating a structure suitable for host cell infiltration and remodeling. 37 In addition, while a solution of percutaneously injected ECM components is likely to be rapidly absorbed, the same components in gel state are likely to display greater durability and persist for the duration of tendon repair. The long-term objective of this work is to develop a gel from decellularized human tendon that is rich in tendon ECM components, durable, biocompatible and immunologically inert. A gel with these properties may augment tendon repair and accelerate tendon healing. The present study describes a method to solubilize human decellularized tendon and to repolymerize the soluble tendon ECM under physiological conditions. This work characterizes the gel content, gelation kinetics, rheological properties in vitro, and in vivo biocompatibility of the gel in an animal model. Cell interaction with multipotent adipoderived stem cells (ASCs) is also addressed in vitro to rule out possible cytotoxicity.
Page 7 of 46
Tissue Engineering Part A Design and Characterization of an Injectable Tendon Hydrogel: A Scaffold for Guided Tissue Regeneration in the Musculoskeletal System (doi: 10.1089/ten.TEA.2013.0207) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
7 Materials and methods: 1. Tissue decellularization and material processing Human flexor digitorum profundus (FDP), flexor digitorum superficialis (FDS) and flexor pollicis longus (FPL) tendons were harvested from fresh-frozen cadaveric forearms (Science Care, Phoenix, Arizona). Epitenon, synovial sheath, and muscule tissue were meticulously debrided. Distally, FDS tendons were transected 2cm proximal to the chiasma, and FDP and FPL tendons were transected 1cm proximal to the osteotendinous junction. The tendons were then decellularized following a previously reported protocol 46. In brief, scaffolds were treated with 0.1% ethylenediaminetetraacetic acid (EDTA) for 4 hours followed by 0.1% sodium dodecyl sulfate (SDS) in 0.1% EDTA for 24 hours at room temperature with constant agitation. Scaffolds were washed in PBS and stored at -80oC. The frozen decellularized material was lyophilized, and milled into a fine powder using a Wiley Mini Mill (Thomas Scientific, Sedesboro, NJ, USA). The powder was stored at 4 ◦C until needed for use. A DNA assay along with routine H&E histology and SYTO green-fluorescent nucleic acid staining was used to quantify the effectiveness of decellularization of tendons. This is described in detail in a previous publication. 46 In short, DNA was extracted from lyophilized tendons using a DNeasy kit (QIAGEN). The concentration of the extract was determined using an ultra- violet spectrophotometer (Biophotometer 22331; Eppendorf) at a wavelength of 260 nm. The concentration of the samples was calculated by software on the spectrophotometer using a known extinction coefficient for dsDNA.
2. ECM gel formation The extracellular matrix material was enzymatically digested by adding a 1 mg/ml solution of pepsin (Sigma, St Louis, MO) in 0.02 M HCl such that the final concentration of material
Page 8 of 46
Tissue Engineering Part A Design and Characterization of an Injectable Tendon Hydrogel: A Scaffold for Guided Tissue Regeneration in the Musculoskeletal System (doi: 10.1089/ten.TEA.2013.0207) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
8 was 10, 20 and 30 mg/ml (1-3% respectively, dry weight). Optimal pH for pepsin digestion was explored and set to 2.2 47 Increasing molarity of HCl is needed to accomplish optimal digestion with increasing concentration of tendon powder (see Table 1 in supplements). The material was digested for up to 72h (12, 24, 36, 48 and 72h) at room temperature with constant stirring. The liquid was checked for pH and homogeneity under a microscope every 12h to optimize the conditions for digestion. For the in vivo experiments and reseeding of 2% gels in vitro, the digestion time was set to 24h to ensure complete digestion. While cooled on ice, the pH was neutralized to a pH of 7.4 and salt concentration was adjusted with the addition of 0.2M NaOH (1/10 of original digest volume) and 10x PBS (1/10 of final neutralized volume). The final mixture was then allowed to gel for 20-60 minutes at 37°C. Gelation was confirmed macroscopically and with rheology (see below and Figure 1). For in vitro reseeding of the gel, pepsin activity was completely reversed at pH >8 before the final solution pH was set at 7.4. The ECM solution was stored as neutralized samples with a pH 7.4, and as acidic samples with a pH 2.2 under different conditions (4°, -80°and lyophilized). The acidic samples were neutralized after storage, but prior to gelation at 37°. Gelation was observed on day 1, 7, and 10 over 30 minutes and recorded as (-) no gelation, (+/-) weak gelation, (+) gelation.
3. Evaluation methods Mass Spectrometry Samples were prepared using a FASP (Filter aided proteome preparation) 48 protocol, where the sample was solubilized in SDS, DTT and Tris-HCL and digested overnight by trypsin. The peptides were loaded onto a self-packed fused silica C18 analytical column, interfaced by a Bruker Michrom Advance (Auburn, CA, USA) source with a flow rate of 600 nL/min and a spray voltage of 1.7kV. The mass spectrometer was a LTQ Orbitrap Velos, Thermo
Page 9 of 46
Tissue Engineering Part A Design and Characterization of an Injectable Tendon Hydrogel: A Scaffold for Guided Tissue Regeneration in the Musculoskeletal System (doi: 10.1089/ten.TEA.2013.0207) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
9 Scientific (Vancouver, Canada), set in data dependent acquisition mode fragmenting the top 12 most intense precursor ions. The RAW data was converted to mzXML format and database searched against the human uniprot-sprot database using Sequest on a Sorcerer platform. The data was visualized using Scaffold 3, Proteome Software (Portland, OR, USA).
Rheology: storage modulus, temperature characteristics Rheological measurements were made using a TA Instruments ARG2 Rheometer. Two parallel steel plates (40 mm diameter) at 100μm gap height were used on 500 μl gels under the different conditions after 24 h of gelation. The setup was such that the sample completely filled the gap between the rheology plates. For each condition, the storage modulus (G’) and loss modulus (G’’) over frequencies of 0.1-10 rad s-1 (frequency sweep) were recorded for every gel condition in triplicate and plotted. To document gelation properties at increasing temperature (temperature sweep), the peltier probes of the rheometer were preheated to 25°C. Measurements of each sample were recorded at 1 rad sec-1 at increasing temperature, 2°C per minute until 37°C was reached. Temperature was maintained at 37°C for 24 minutes until the gel had polymerized and the modulus stabilized.
Scanning electron microscopy Samples were fixed from 24hrs to 4 days at 4˚C with 4% paraformaldehyde and 2% glutaraldehyde in 0.1M sodium cacodylate buffer (pH 7.2), rinsed in the same buffer and post-fixed for 1hr with 1% aqueous OsO4. After dehydration in an ascending ethanol series (50, 70, 90, 100 %; 15min each) samples were dried with liquid CO2 in a Tousimis Autosamdri-815B apparatus (Tousimis, Rockville, MD, USA), mounted on adhesive copper tape on 15mm aluminum stubs (Ted Pella, Redding, CA), and sputter-coated with 50A of
Page 10 of 46
Tissue Engineering Part A Design and Characterization of an Injectable Tendon Hydrogel: A Scaffold for Guided Tissue Regeneration in the Musculoskeletal System (doi: 10.1089/ten.TEA.2013.0207) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
10 Au/Pd using a Denton DeskII Sputter Coater. Visualization was performed with a Hitachi S3400N Variable Pressure (VP) SEM operated at 15kV, working distance 7-8mm, and secondary electron (SE) detection under high-vacuum conditions (
Tissue Engineering Part A Design and Characterization of an Injectable Tendon Hydrogel: A Scaffold for Guided Tissue Regeneration in the Musculoskeletal System (doi: 10.1089/ten.TEA.2013.0207) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
1
Design and Characterization of an Injectable Tendon Hydrogel: A Novel Scaffold for Guided Tissue Regeneration in the Musculoskeletal System
Simon Farnebo1, Colin Y Woon1, Taliah Schmitt1, Lydia-Marie Joubert 2, Max Kim1, Hung Pham1, James Chang1
1
Division of Plastic and Reconstructive Surgery, Stanford University Medical Center
and Section of Plastic Surgery, VA Palo Alto Health Care System 2
Cell Sciences Imaging Facility, Stanford University Medical School, Stanford.
Keywords: Tendon, ECM (extracellular matrix), hydrogel, tissue engineering, scaffold
Corresponding author: James Chang MD Division of Plastic and Reconstructive Surgery Stanford University Medical Center 770 Welch Road, Suite 400 Stanford, CA 94304 [email protected]
Page 2 of 46
Tissue Engineering Part A Design and Characterization of an Injectable Tendon Hydrogel: A Scaffold for Guided Tissue Regeneration in the Musculoskeletal System (doi: 10.1089/ten.TEA.2013.0207) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
2 Abstract: A biocompatible hydrogel consisting of extracellular matrix (ECM) from human tendons is described as a potential scaffold for guided tissue regeneration and tissue engineering purposes. Lyophilized decellularized tendons were milled and enzymatically digested to form an ECM solution. The ECM solution properties are assessed by proteome analysis with mass spectrometry, and the material’s rheological properties are determined both as a function of frequency, temperature and time. In vivo application of the gel in a rat model is assessed for remodeling and host cell repopulation. Histology for macrophage invasion, fibroblast repopulation and nanoscale properties of the gel is assessed. Gel interaction with multipotent adipoderived stem cells (ASCs) is also addressed in vitro to assess possible cytotoxicity and its ability to act as a delivery vehicle for cells. Proteome analysis of the ECM-solution and gel mass spectroscopy identified the most abundant 150 proteins, of which two isoforms of Collagen I represented more than 55% of the sample. Rheology showed that storage (G’) and loss (G”) of the ECM solution were stable at room temperature but displayed sigmoidal increases after approximately 15 minutes at 37°C, matching macroscopic observations of its thermo responsiveness. G’ and G” of the gel at 1 rad/s were 213.1±19.9 Pa and 27.1±2.4 Pa respectively. Electron microscopy revealed fiber alignment and good structural porosity in the gel, as well as invasion of cells in vivo. Histology also showed early CD 68+ macrophage invasion throughout the gel, followed by increasing numbers of fibroblast cells. ASCs mixed with the gel in vitro proliferated, indicating good biocompatibility. This ECM solution can be delivered percutaneously into a zone of tendon injury. After injection, the thermoresponsive behavior of the ECM solution allows it to polymerize and form a porous gel at body temperature. A supportive nanostructure of collagen fibers is
Page 3 of 46
Tissue Engineering Part A Design and Characterization of an Injectable Tendon Hydrogel: A Scaffold for Guided Tissue Regeneration in the Musculoskeletal System (doi: 10.1089/ten.TEA.2013.0207) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
3 established that conforms to the three dimensional space of the defect. This hydrogel holds the distinctive composition specific for tendon ECM, where tissue specific cues facilitate host cell infiltration and remodeling. The results presented indicate that injectable ECM materials from tendon may offer a promising alternative in the treatment of tendinopathies and acute tendon injuries.
Page 4 of 46
Tissue Engineering Part A Design and Characterization of an Injectable Tendon Hydrogel: A Scaffold for Guided Tissue Regeneration in the Musculoskeletal System (doi: 10.1089/ten.TEA.2013.0207) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
4 Introduction: Ligament and tendon healing are important aspects of orthopedic surgery, hand surgery and sports medicine. In tendinopathy, tendon overuse leads to significant histological and biochemical changes that alter biomechanical and material properties 1, causing accumulative microscopic tears that ultimately result in full-thickness tears 2. These injuries are most commonly seen in the rotator cuff, Achilles tendon, quadriceps tendon, and patella tendon. In addition, medial and lateral epicondylitis of the elbow, and other chronic enthesopathies (disorders of the tendon-bone attachment), are believed to involve similar mechanisms of injury. Microstructural changes seen include micro-tears, thinning and disorganization of collagen fibers 3, 4, neovascularization of the tendon matrix 5, and morphological changes to tenocytes 6. Methods attempted by various authors to augment and stimulate healing 7 include injections of platelet rich plasma (PRP) 8 9 10, whole blood 11, or growth hormones 11-13; and the addition of multipotent stem cells. 14-16 Ligament and tendon healing may be accelerated by direct delivery of biocompatible scaffolds to the zone of injury that would facilitate in situ regeneration by means of threedimensional (3d) guided tissue regeneration 17. The 3d-scaffold would support the physical properties of the tissue and facilitate migration of surrounding cells into the scaffold microenvironment. These cells would subsequently proliferate and replace the scaffold with regenerated tissue. The application of a biodegradable scaffold is attractive as it may act both as a structural matrix and as a three-dimensional carrier of cells or growth hormones. 18 19 20 21
Most of the investigated injectable hydrogels have been synthetic polymers 22-24 with defined structural, chemical, and mechanical properties designed for a desired application, or composed of individual components of the ECM. Gels composed of type I collagen 25-27, hyaluronic acid 28, 29, or laminin 30-32 display desirable biocompatibility and strength.
Page 5 of 46
Tissue Engineering Part A Design and Characterization of an Injectable Tendon Hydrogel: A Scaffold for Guided Tissue Regeneration in the Musculoskeletal System (doi: 10.1089/ten.TEA.2013.0207) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
5 However, when taken out of their ECM context, they do not appropriately match the injured microenvironment and therefore lose some of the essential cues that would be needed for cellular repopulation. There have been efforts to develop injectable hydrogels that are derived from the tissue being treated or replaced. These gels would be a better match as they would possess an ECM composition matched to the native tissue, as well as the chemical and biological cues inherent to these matrix components 33 34 35 36 37 38 39 . A hydrogel based on decellularized materials will thus have a distinct composition that closely matches the tissue of origin, 40 34 allowing for the development of tissue specific scaffolds for appropriate cell–matrix interactions. Specific to tendon repair and regeneration, collagen fibril segments (CFS) are tendon ECM elements used in fetal tendon development 41, 42. Numerous studies suggest that intrinsic tendon repair uses recycled CFS as an immediate source in reconnecting severed fibrils during tendon healing 42-45, and that endogenous tendon fibroblasts will bridge the gap between the severed tendon ends with the deposition of new connective tissue matrix as part of an intrinsic tendon repair. It is proposed that released CFS from recycled collagen fibers could generate a pool of CFS that supply a portion of the building blocks utilized at the healing site 42, 44. These studies do not directly address the issue of ECM element placement and delivery. Directed delivery of ECM elements using needle injection, or implantation during surgery, or using delivery vehicles such as hydrogel-impregnated collagen sponges or hydrogel-coated sutures may address the two-fold objective of providing native materials, and placing ECM components, such as collagen fibril segments (CFS) from recycled collagen fibers directly at the repair site, where active ECM synthesis is taking place. Although type I collagen is the major ECM element involved, it is possible that other
Page 6 of 46
Tissue Engineering Part A Design and Characterization of an Injectable Tendon Hydrogel: A Scaffold for Guided Tissue Regeneration in the Musculoskeletal System (doi: 10.1089/ten.TEA.2013.0207) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
6 ECM components are involved in repair and an ECM-rich solution composed of tendon digestate may augment tendon healing to an extent greater than purified CFS alone. An ECM solution with appropriate viscosity can be delivered in a minimally invasive fashion by injection, and then conform to the three-dimensional space following deposition. It has been shown previously that the thermoresponsive behavior of different ECM solutions allows it to polymerize and form a gel at body temperature, creating a structure suitable for host cell infiltration and remodeling. 37 In addition, while a solution of percutaneously injected ECM components is likely to be rapidly absorbed, the same components in gel state are likely to display greater durability and persist for the duration of tendon repair. The long-term objective of this work is to develop a gel from decellularized human tendon that is rich in tendon ECM components, durable, biocompatible and immunologically inert. A gel with these properties may augment tendon repair and accelerate tendon healing. The present study describes a method to solubilize human decellularized tendon and to repolymerize the soluble tendon ECM under physiological conditions. This work characterizes the gel content, gelation kinetics, rheological properties in vitro, and in vivo biocompatibility of the gel in an animal model. Cell interaction with multipotent adipoderived stem cells (ASCs) is also addressed in vitro to rule out possible cytotoxicity.
Page 7 of 46
Tissue Engineering Part A Design and Characterization of an Injectable Tendon Hydrogel: A Scaffold for Guided Tissue Regeneration in the Musculoskeletal System (doi: 10.1089/ten.TEA.2013.0207) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
7 Materials and methods: 1. Tissue decellularization and material processing Human flexor digitorum profundus (FDP), flexor digitorum superficialis (FDS) and flexor pollicis longus (FPL) tendons were harvested from fresh-frozen cadaveric forearms (Science Care, Phoenix, Arizona). Epitenon, synovial sheath, and muscule tissue were meticulously debrided. Distally, FDS tendons were transected 2cm proximal to the chiasma, and FDP and FPL tendons were transected 1cm proximal to the osteotendinous junction. The tendons were then decellularized following a previously reported protocol 46. In brief, scaffolds were treated with 0.1% ethylenediaminetetraacetic acid (EDTA) for 4 hours followed by 0.1% sodium dodecyl sulfate (SDS) in 0.1% EDTA for 24 hours at room temperature with constant agitation. Scaffolds were washed in PBS and stored at -80oC. The frozen decellularized material was lyophilized, and milled into a fine powder using a Wiley Mini Mill (Thomas Scientific, Sedesboro, NJ, USA). The powder was stored at 4 ◦C until needed for use. A DNA assay along with routine H&E histology and SYTO green-fluorescent nucleic acid staining was used to quantify the effectiveness of decellularization of tendons. This is described in detail in a previous publication. 46 In short, DNA was extracted from lyophilized tendons using a DNeasy kit (QIAGEN). The concentration of the extract was determined using an ultra- violet spectrophotometer (Biophotometer 22331; Eppendorf) at a wavelength of 260 nm. The concentration of the samples was calculated by software on the spectrophotometer using a known extinction coefficient for dsDNA.
2. ECM gel formation The extracellular matrix material was enzymatically digested by adding a 1 mg/ml solution of pepsin (Sigma, St Louis, MO) in 0.02 M HCl such that the final concentration of material
Page 8 of 46
Tissue Engineering Part A Design and Characterization of an Injectable Tendon Hydrogel: A Scaffold for Guided Tissue Regeneration in the Musculoskeletal System (doi: 10.1089/ten.TEA.2013.0207) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
8 was 10, 20 and 30 mg/ml (1-3% respectively, dry weight). Optimal pH for pepsin digestion was explored and set to 2.2 47 Increasing molarity of HCl is needed to accomplish optimal digestion with increasing concentration of tendon powder (see Table 1 in supplements). The material was digested for up to 72h (12, 24, 36, 48 and 72h) at room temperature with constant stirring. The liquid was checked for pH and homogeneity under a microscope every 12h to optimize the conditions for digestion. For the in vivo experiments and reseeding of 2% gels in vitro, the digestion time was set to 24h to ensure complete digestion. While cooled on ice, the pH was neutralized to a pH of 7.4 and salt concentration was adjusted with the addition of 0.2M NaOH (1/10 of original digest volume) and 10x PBS (1/10 of final neutralized volume). The final mixture was then allowed to gel for 20-60 minutes at 37°C. Gelation was confirmed macroscopically and with rheology (see below and Figure 1). For in vitro reseeding of the gel, pepsin activity was completely reversed at pH >8 before the final solution pH was set at 7.4. The ECM solution was stored as neutralized samples with a pH 7.4, and as acidic samples with a pH 2.2 under different conditions (4°, -80°and lyophilized). The acidic samples were neutralized after storage, but prior to gelation at 37°. Gelation was observed on day 1, 7, and 10 over 30 minutes and recorded as (-) no gelation, (+/-) weak gelation, (+) gelation.
3. Evaluation methods Mass Spectrometry Samples were prepared using a FASP (Filter aided proteome preparation) 48 protocol, where the sample was solubilized in SDS, DTT and Tris-HCL and digested overnight by trypsin. The peptides were loaded onto a self-packed fused silica C18 analytical column, interfaced by a Bruker Michrom Advance (Auburn, CA, USA) source with a flow rate of 600 nL/min and a spray voltage of 1.7kV. The mass spectrometer was a LTQ Orbitrap Velos, Thermo
Page 9 of 46
Tissue Engineering Part A Design and Characterization of an Injectable Tendon Hydrogel: A Scaffold for Guided Tissue Regeneration in the Musculoskeletal System (doi: 10.1089/ten.TEA.2013.0207) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
9 Scientific (Vancouver, Canada), set in data dependent acquisition mode fragmenting the top 12 most intense precursor ions. The RAW data was converted to mzXML format and database searched against the human uniprot-sprot database using Sequest on a Sorcerer platform. The data was visualized using Scaffold 3, Proteome Software (Portland, OR, USA).
Rheology: storage modulus, temperature characteristics Rheological measurements were made using a TA Instruments ARG2 Rheometer. Two parallel steel plates (40 mm diameter) at 100μm gap height were used on 500 μl gels under the different conditions after 24 h of gelation. The setup was such that the sample completely filled the gap between the rheology plates. For each condition, the storage modulus (G’) and loss modulus (G’’) over frequencies of 0.1-10 rad s-1 (frequency sweep) were recorded for every gel condition in triplicate and plotted. To document gelation properties at increasing temperature (temperature sweep), the peltier probes of the rheometer were preheated to 25°C. Measurements of each sample were recorded at 1 rad sec-1 at increasing temperature, 2°C per minute until 37°C was reached. Temperature was maintained at 37°C for 24 minutes until the gel had polymerized and the modulus stabilized.
Scanning electron microscopy Samples were fixed from 24hrs to 4 days at 4˚C with 4% paraformaldehyde and 2% glutaraldehyde in 0.1M sodium cacodylate buffer (pH 7.2), rinsed in the same buffer and post-fixed for 1hr with 1% aqueous OsO4. After dehydration in an ascending ethanol series (50, 70, 90, 100 %; 15min each) samples were dried with liquid CO2 in a Tousimis Autosamdri-815B apparatus (Tousimis, Rockville, MD, USA), mounted on adhesive copper tape on 15mm aluminum stubs (Ted Pella, Redding, CA), and sputter-coated with 50A of
Page 10 of 46
Tissue Engineering Part A Design and Characterization of an Injectable Tendon Hydrogel: A Scaffold for Guided Tissue Regeneration in the Musculoskeletal System (doi: 10.1089/ten.TEA.2013.0207) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
10 Au/Pd using a Denton DeskII Sputter Coater. Visualization was performed with a Hitachi S3400N Variable Pressure (VP) SEM operated at 15kV, working distance 7-8mm, and secondary electron (SE) detection under high-vacuum conditions (
