Patent Application
20240299622
The present invention relates generally to biphasic osteotendinous repair scaffolds, and more particularly to biphasic osteotendinous repair scaffolds comprising a first phase comprising electrochemically aligned collagen (ELAC) threads and a calcium phosphate mineral and having a major surface, and a second phase comprising ELAC threads and having a major surface.
Soft tissues such as ligaments or tendons often rupture at their interface with bone. Attachment of soft tissues to bone footprint is challenging for various reasons. First, the soft tissues retract and re-apposing them on the bone footprint may require too much tension. In this context, a bridging material to traverse that gap would be helpful. Second, soft tissues have evolved such that molecules in their biological compositions prevent mineral crystals from growing. Soft tissues thus integrate with bone tissue very slowly, through formation of a scar tissue which slowly and only partially remodels into a cohesive bone-soft tissue transition. This is why reattachment of soft tissue such as ligaments or tendons to bone result in re-ruptures on a high percentage of patients. These failures can occur in many locations in the body including, among others, the rotator cuff (RC) at the shoulder, Achilles tendon at the heel, and ligaments of the knee.
The social and economic burden of rotator cuff (RC) defects is highly significant. Tendons and muscles of the RC complex are critical to movement of arms. Full or partial thickness tears of RC occur because of acute trauma, repetitive occupational use, or degeneration during aging. Essential functions such as reaching out to shelves or tooth-brushing are compromised along with impairment in the quality of life (7, 8). There are 1.2 million physician visits for RC problems annually, and 330,000 RC procedures/year in the U.S. (98 per 100,000) at an estimated economic burden of $3.8 billion in 2019 (9).
There is an unmet need for effective treatment of patients with irreparable RC tears. Early stage small-sized tears can be treated by rest, physical therapy, or cortisol injections to provide symptomatic relief (10). Mid- to large tears are generally repaired by arthroscopic surgery to reattach torn segment to bone footprint on the humeral head by suture anchors (11). Some large tears may require overlay reinforcement patches (allogeneic, xenogeneic, or polymeric); however, their efficacy is not well-proven because evidence comes from lower-level case series generally with limited sample sizes (12).
Our interest lies in the repair of irreparable tears wherein muscle atrophy and fatty infiltration is pronounced, and the musculotendinous complex is retracted notably, making it impossible to perform primary repair by opposing the tendon on the bone-footprint without creating a damaging level of tension that would result in suture pull-out. This is an unmet need whose fulfillment requires a gap bridging biomaterial that will also connect the remnant musculotendinous complex to bone-footprint.
Subacromial spacers, such as the INSPACE balloon device, have found some acceptance in providing some relief, but a very recent double-blind, randomized and multi-center clinical trial concluded that the INSPACE balloon device was not more effective than debridement alone (13). Otherwise, irreparable tears remain outside the domain of arthroscopic surgery and often call for open repair.
There are no off-the-shelf biomaterial solutions that address irreparable tears.
Autografts do not have widespread use in repair due to lack of suitably sized donor locations. Allografts and xenografts may elicit immune response, scar tissue formation, or disease transmission (12, 14). Decellularized grafts also lack a connected open pore structure. Thus, cellular infiltration is highly restricted. Some patients are treated by invasive muscle transfer procedures (15). Otherwise, most patients live with limited mobility and chronic pain, eventually undergoing total joint arthroplasty (1, 2).
A regenerative platform for treatment of irreparable tears needs to fulfill the following: 1) Bulk tendon regeneration: Muscle retraction results in a gap between tendon and bone which needs to be bridged by off-the-shelf product that should drive regeneration of neo-tendon. 2) Surgical Handling: The scaffold should retain sutures to be attached to tendon proximally and to bone distally. 3) In vivo durability: The scaffold should bear functional loads in vivo. 4) Integration with bone: The distal end of the scaffold should be osteoconductive to accommodate bony ingrowth.
Monophasic scaffolds do not address the chronic RC degeneration problem adequately. Existing strategies generally address either bone or tendon aspects, whereas a chronic degeneration situation requires reconstitution of both domains. The delivery of osteoinductive factors to bone-tendon interface, for example, injectable calcium phosphates (16, 17), collagen gel/sponge/paste (18-20), etc., have reportedly resulted in increased strength and stiffness (18-21), and increased ossification (17, 22). Delivery of cytokines that promote connective tissue, such as GDF5 (23), CDMP2 (24), TGFβ 1-3 (25-28), and FGFs (28-31), have been reported to increase strength (23, 24, 26, 31), collagen output (23, 25), or collagen fiber organization (25, 27, 31). There are limitations to these studies, though, including the following: 1) short follow up period (<4 weeks), 2) beneficial outcomes not sustained at longer time points, 3) lack of characterization of tendon-specific markers, such as scleraxis or tenomodulin, for assessment of endpoint healing, and 4) generally neither a gap is imposed nor a chronic degeneration is induced. They do not model the irreparable condition sufficiently.
Moreover, to the best of our knowledge, only two research studies have used multiphasic scaffolds in vivo for RC repair; and only in an acute tear context as an overlay patch (47) or as a sheet (48) insertion between bone and tendon. These approaches are useful to treat acute tears where native tendon bears the load. However, chronic degeneration requires full load bearing scaffolds that address both tendon and bone regeneration.
Thus, a need exists for a bioinductive scaffold for regenerating bulk tendon volume to enable patients to sustain daily necessities of life.
A biphasic osteotendinous repair scaffold is disclosed. The biphasic osteotendinous repair scaffold comprises a first phase comprising electrochemically aligned collagen (ELAC) threads and a calcium phosphate mineral and having a major surface. The biphasic osteotendinous repair scaffold also comprises a second phase comprising ELAC threads and having a major surface. The ELAC threads of the first phase are braided and crosslinked. The ELAC threads of the second phase are braided and crosslinked. The ELAC threads of the first and second phases form first and second interconnected macroporosities throughout the first and second phases, respectively. The calcium phosphate mineral of the first phase is distributed on the major surface of the first phase and within the interconnected macroporosity of the first phase. The second phase is adjacent to the first phase.
In some embodiments, the ELAC threads of the first phase are twisted to form a yarn, the ELAC threads of the second phase are twisted to form a yarn, the yarn of the first phase is braided, thereby forming the ELAC threads of the first phase that are braided, and the yarn of the second phase is braided, thereby forming the ELAC threads of the second phase that are braided. In some of these embodiments, the yarn of the first phase comprises a 4-ply yarn and a 2-ply yarn, the 4-ply yarn of the first phase is used as triaxial threads within the first phase, and the 2-ply yarn of the first phase is used as oblique threads within the first phase, and the yarn of the second phase comprises a 4-ply yarn and a 2-ply yarn, the 4-ply yarn of the second phase is used as triaxial threads within the second phase, and the 2-ply yarn of the second phase is used as oblique threads within the second phase. Also in some of these embodiments, three or more concentric layers of the yarn of the first phase are braided on top of each other successively, and three or more concentric layers of the yarn of the second phase are braided on top of each other successively.
In some embodiments, the ELAC threads of the first phase are braided as monofilaments, and the ELAC threads of the second phase are braided as monofilaments.
In some embodiments, the scaffold has a load-bearing axis, and a majority of the ELAC threads are positioned along the load-bearing axis.
In some embodiments, the ELAC threads of the first and second phases are crosslinked with an iridoid crosslinking agent. In some of these embodiments, the iridoid crosslinking agent comprises one or more of genipin, loganin aglycone, oleuropein aglycone, or E-6-O-methoxycinnamoyl scandoside methyl ester aglycone. In some of these embodiments, the iridoid crosslinking agent comprises genipin.
In some embodiments, the calcium phosphate mineral comprises osteoinductive hydroxyapatite crystals or dicalcium phosphate dihydrate crystals.
In some embodiments, the second phase is adjacent to the first phase based on being contiguous with the first phase.
In some embodiments, the second phase is adjacent to the first phase based on being attached directly or indirectly to the first phase.
In some embodiments, the scaffold further comprises mesenchymal stem cells seeded on the ELAC threads of the first phase. In some of these embodiments, the mesenchymal stem cells are autologous mesenchymal stem cells. Also in some of these embodiments, to the extent that human or animal cells other than mesenchymal stem cells are present on the ELAC threads of the first phase, more mesenchymal stem cells are present than the other human or animal cells.
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The biphasic osteotendinous repair scaffold disclosed herein, having a structure comprising two adjacent phases, should provide an effective treatment for patients with irreparable RC tears.
An exemplary biphasic osteotendinous repair scaffold 100 is shown in
The biphasic osteotendinous repair scaffold 100 comprises a first phase 102 comprising electrochemically aligned collagen (ELAC) threads 104 and a calcium phosphate mineral 106 and having a major surface 108, and a second phase 110 comprising ELAC threads 112 and having a major surface 114. The ELAC threads 104 of the first phase 102 are braided and crosslinked. The ELAC threads 112 of the second phase 110 also are braided and crosslinked. The ELAC threads 104, 112 of the first and second phases 102, 110 form first and second interconnected macroporosities 116, 118 throughout the first and second phases 102, 110, respectively. The calcium phosphate mineral 106 of the first phase 102 is distributed on the major surface 108 of the first phase 102 and within the interconnected macroporosity 116 of the first phase 102. The second phase 110 is adjacent to the first phase 102.
The first phase 102 is macroporous and mineralized prior to implantation into a patient (also termed pre-mineralized) to accommodate and promote ingrowth of bone at the major surface 108 of the first phase 102. The second phase 110 is macroporous to accommodate ingrowth of tendon at the major surface 114 of the second phase 110. As noted, the second phase 110 is adjacent to the first phase 102, for example based on being contiguous with the first phase 102, or being attached directly or indirectly to the first phase 102. During use, the first phase 102 is put in contact with bone of the patient at the major surface 108 of the first phase 102 by implantation. The second phase 110 is positioned to allow ingrowth of tendon. The duality of the structure of the biphasic osteotendinous repair scaffold 100 allows for hard tissue growth at the major surface 108 of the pre-mineralized first phase 102 of the biphasic osteotendinous repair scaffold 100 and soft tissue growth at the major surface 114 of the unmineralized second phase 110 of the biphasic osteotendinous repair scaffold 100, integrating the disparate hard and soft tissue types effectively.
In some embodiments, the calcium phosphate mineral 106 comprises osteoinductive hydroxyapatite crystals or dicalcium phosphate dihydrate crystals. As noted above, the first phase 102 of the biphasic osteotendinous repair scaffold 100 comprises the calcium phosphate mineral 106. The calcium phosphate mineral 106 can be formed as osteoinductive crystals, for example, osteoinductive hydroxyapatite crystals or dicalcium phosphate dihydrate crystals. This can be accomplished by repeated sequential soaking of a portion of a scaffold in a solution of calcium and a solution of phosphate. The molar ratios of the calcium and the phosphate in the solutions can be varied to determine the type of crystals formed.
The ELAC threads 104 of the first phase 102 and the ELAC threads 112 of the second phase 110 can be used directly as monofilaments and/or as a multi-ply yarns formed from multiple monofilaments, for example, 2-ply, 3-ply, 4-ply, or 5-ply yarns, formed from 2, 3, 4, or 5 monofilaments, respectively.
In some embodiments, the second phase 110 is contiguous with the first phase 102. In accordance with these embodiments, a biphasic osteotendinous repair scaffold 100 can be made by the following process. First, an initial uncrosslinked scaffold is formed from ELAC threads. For example, yarn can be formed from the ELAC threads. The yarn can be braided over a carrier mandrel, with two or more concentric layers, e.g., three concentric layers, being braided on top of each other successively. The braids can be cut into segments and flattened as a ribbon, e.g., each segment having a thickness, for example, of 1 to 5 mm, a width, for example, of 5 to 50 mm, and a length, for example, of 20 to 60 mm. These dimensions would be suitable for rotator cuff repair in human patients. These dimensions and others may be suitable for other applications too. Both ends of a segment can be fused with an acidic collagen solution to obtain the initial uncrosslinked scaffold. The initial uncrosslinked scaffold can then be treated with a crosslinking agent, such genipin, to form a crosslinked scaffold. Then a first portion of the crosslinked scaffold can be soaked in two mineralizing solutions, one being a calcium solution and the other being a phosphate solution. Repeated sequential soaking of the first portion in these two solutions with intermediate drying steps results in ongrowth and ingrowth of calcium phosphate mineral, for example hydroxyapatite crystals or dicalcium phosphate dihydrate crystals, in the first portion of the crosslinked scaffold that is dipped in the solutions, thereby forming the first phase 102. A second remaining portion of the crosslinked scaffold that is not soaked in the solutions remains unmineralized, and corresponds to the second phase 110. By this process, a single continuum of a biphasic osteotendinous repair scaffold 100 is obtained, a first phase 102 that is mineralized and a second phase 110 that is unmineralized, wherein the second phase 110 is contiguous with the first phase 102.
In some embodiments, the second phase 110 is attached to the first phase 102 based on being stitched or adhered to the first phase 102.
In some embodiments, the first phase 102 can be induced along end-to-end segments, and thus serially connected. The mineralized end of the series can then be inserted into a pocket that is machined in bone footprint and secured by a pin, sutures, or other methods for native bone to integrate.
Considering yarn formed from ELAC threads in more details, in some embodiments the ELAC threads 104 of the first phase 102 are twisted to form a yarn 120, the ELAC threads 112 of the second phase 110 are twisted to form a yarn 122, the yarn 120 of the first phase 102 is braided, thereby forming the ELAC threads 104 of the first phase 102 that are braided, and the yarn 122 of the second phase 110 is braided, thereby forming the ELAC threads 112 of the second phase 110 that are braided.
In some of these embodiments, the yarn 120 of the first phase 102 comprises a 4-ply yarn and a 2-ply yarn, the 4-ply yarn of the first phase 102 is used as triaxial threads 124 within the first phase 102, and the 2-ply yarn of the first phase 102 is used as oblique threads 126 within the first phase 102. Also in these embodiments, the yarn 122 of the second phase 110 comprises a 4-ply yarn and a 2-ply yarn, the 4-ply yarn of the second phase 110 is used as triaxial threads 128 within the second phase 110, and the 2-ply yarn of the second phase 110 is used as oblique threads 130 within the second phase 110.
Also in some of these embodiments, three or more concentric layers of the yarn 120 of the first phase 102 are braided on top of each other successively, and three or more concentric layers of the yarn 122 of the second phase 110 are braided on top of each other successively.
Alternatively or additionally, in some of these embodiments a single layer long braid can be folded to obtain a multilayer scaffold.
In some embodiments, the ELAC threads 104 of the first phase 102 are braided as monofilaments, and the ELAC threads 112 of the second phase 110 are braided as monofilaments.
As noted above, the biphasic osteotendinous repair scaffold 100 comprises a first phase 102 having a major surface 108 and a second phase 110 has a major surface 114. The major surfaces 108, 114 of the first and second phases 102, 110 can be, for example, top, bottom, and/or side surfaces of the biphasic osteotendinous repair scaffold 100.
In some embodiments, the first phase 102 forms a first layer having a major surface, the second phase 110 forms a second layer having a major surface, and the first layer is attached to the second layer across their respective major surfaces, thereby forming a bilayer. In accordance with this embodiment, the biphasic osteotendinous repair scaffold 100 can be implanted, for example, between bone and tendon, with the first layer facing the bone and the second layer facing the tendon. The biphasic osteotendinous repair scaffold 100 can then act as an intermediate layer between the bone and tendon and thereby serve its function.
In some embodiments, the biphasic osteotendinous repair scaffold 100 has a load-bearing axis 132, and a majority of the ELAC threads 104, 112 are positioned along the load-bearing axis 132.
In some embodiments, the ELAC threads 104, 112 of the first and second phases 102, 110 are crosslinked with an iridoid crosslinking agent. In some of these embodiments, the iridoid crosslinking agent comprises one or more of genipin, loganin aglycone, oleuropein aglycone, or E-6-O-methoxycinnamoyl scandoside methyl ester aglycone. In some of these embodiments, the iridoid crosslinking agent comprises genipin.
As noted above, the ELAC threads 104 of the first phase 102 are braided and crosslinked, and the ELAC threads 112 of the second phase 110 also are braided and crosslinked. In some embodiments, the ELAC threads 104, 112 are first braided, then crosslinked. In some embodiments, the ELAC threads 104, 112 are first crosslinked, then braided.
In some cases it may be advantageous to seed the biphasic osteotendinous repair scaffold 100 with mesenchymal stem cells to promote ongrowth and ingrowth of bone at the major surface 108 of the first phase 102. Thus, in some embodiments the biphasic osteotendinous repair scaffold 100 further comprises mesenchymal stem cells seeded on the ELAC threads 104 of the first phase 102. In some of these embodiments, the mesenchymal stem cells are autologous mesenchymal stem cells, i.e., mesenchymal stem cells derived from the patient into which the scaffold is to be implanted. Also in some of these embodiments, to the extent that human or animal cells other than mesenchymal stem cells are present on the ELAC threads 104 of the first phase 102, more mesenchymal stem cells are present than the other human or animal cells. For example, in some embodiments, to the extent that human or animal cells other than mesenchymal stem cells are present on the ELAC threads 104 of the first phase 102, at least 51%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% of the total number of human or animal cells present on the ELAC threads 104 of the first phase 102 are macrophages.
We developed our biphasic osteotendinous repair scaffold upon recognizing that monophasic ELAC scaffolds drive tendon-like regeneration in vivo, but provide limited regeneration of the enthesis (5, 6, 46).
For context, the process of electrochemical alignment and compaction (41) generates resilient ELAC threads that can be used to fabricate load-bearing collagen biotextiles (3, 5, 32-38). Type I collagen is the most abundant protein in tendon. Reconstituted collagen gels and sponges are mechanically unfit for replacing load-bearing tendon (39, 40). The ELAC process compacts and aligns collagen molecules as threads whose strength and modulus converge to that of native tendon (36). In the ELAC process, aqueous solution of collagen (medical grade bovine type I, Collagen Solutions Inc., 3 mg/mL) is applied between parallel line electrodes (20-40 V,
Convectional forces induce molecule alignment parallel to the longer axis of threads to a degree comparable to that of tendon (
ELAC-induced cytoskeletal elongation promotes tenogenesis in vitro (3, 37, 43). Tenogenic potential of mesenchymal stem cells increases on anisotropic materials such as decellularized tendon (44), nanofibrous aligned PLLA (45), silk, and also using ELAC threads (
As noted above, monophasic ELAC scaffolds drive tendon-like regeneration in vivo, but provide limited regeneration of the enthesis (5, 6, 46). For example, we applied monophasic woven ELAC scaffolds to repair rabbit infraspinatus (New Zealand White) where 50% of the original tendon was resected distally and repaired. Woven scaffolds were sutured at the proximal end on the remnant tendon, and the distal end to the bone via bone tunnels to bridge the gap defect. Direct repair was included as the positive control where tendon is sharply dissected at enthesis and reattached without segmental defect induction. A gap defect no-repair group was included as the negative control. We assessed regeneration at 6 months (N=11/group, 8 biomechanics and 3 immuno/histology). The result was that Masson's trichrome sections indicated a dense de novo collagen deposition in and around the scaffold in close association with collagen threads (
In mechanical tests involving a monophasic scaffold as presented in
Our biphasic osteotendinous repair scaffold, also termed OsTend scaffold, is intended to address the reconstitution of the enthesis between the scaffold and bone. We will attain a more robust integration at the enthesis by: a) improving the scaffold concept from a monophasic tendon construct to a biphasic osteotendinous repair format by introducing an osteoconductive distal end, b) by adopting braiding instead of weaving as the textile process to position threads predominantly along the load-bearing axis, and c) by modifying the surgical model to nest the osteoconductive end deeper into bone for enhanced anchorage.
Biphasic OsTend scaffold: Spools of collagen threads can be loaded to industrial braiding machinery (Steegers Inc.) to obtain tape-shaped scaffolds with controlled macroporosity for cellular infiltration as shown in (
Osteoconductivity of HA-ELAC: Feasibility has been demonstrated as follows. Osteocalcin expression by mesenchymal stem cells seeded on ELAC is downregulated (
OsTend scaffold was implanted on a segmental defect in rabbit infraspinatus in vivo as described above to assess its osteoconductivity. The osteoconductive distal end was on-laid on the tendon footprint with subchondral bone shaved off to provide access to the marrow stock. We have not inserted the distal end in the humeral head to be able to distinguish mineralizing OsTend scaffold from the native trabecular bone. The same implant was scanned by MicroCT at the baseline, and later at three months after sacrifice. Five equidistant image-sections were obtained from each scan transversely to the longer axis of the osteoconductive segment of the scaffold (
Presently, biomaterials that are used in RC repair clinically are not suitable for irreparable defects. Dermal patches serve a passive mechanical reinforcement function that may be beneficial in augmentation of small to mid-sized tears. However, irreparable tears necessitate regenerative cues that are largely absent in decellularized allografts or xenografts. To the best of our knowledge, there are no biphasic scaffolds in clinical practice that will result in tendon regeneration while integrating with bone effectively.
Our fully-load bearing, defect bridging, suture retaining, structurally multiphasic, compositionally multiphasic, and bioinductively multiphasic regeneration OsTend scaffold disclosed herein offers a way to address the unmet need.
Our OsTend scaffold also may impact other soft-hard tissue regeneration challenges such as ligament-bone, intervertebral disc-endplate, and osteochondral repair.
Our OsTend scaffold's translational potential is significant as well. The manufacturing process can be scaled-up as shown by proof-of-concept braiding run that was accomplished at Steeger Inc. (
Aim 1: Demonstrate that the OsTend scaffold strategy improves repair outcomes in irreparable defect of rotator cuff.
Thread fabrication: Threads will be fabricated as explained above. Four threads will be twisted to obtain a 4-ply yarn to be used as triaxial threads. Two threads will be twisted as 2-ply yarn to be used for oblique threads (
Crosslinking and HA-Infusion: Braiding is performed by using uncrosslinked dry collagen threads. To provide strength in wet state, braided article is crosslinked in 0.625% genipin-ethanol solution for 3 days at room temperature as we described earlier (36). The next step involves growth of HA crystals in the 5 mm long distal segment by alternating soaking (AS) (
Overview and Justification: Rabbit infraspinatus is nominally macroscale (mms in thickness and width, a few centimeters in length). Authoritative reviews on animal models of tendon repair list rabbit in the large animal model group (49). RIC model is also well-established in the prior tendon tissue engineering literature (50, 51). Rabbit RC is similar to human RC in certain regards as elucidated by others (52). Finally, rabbit model offers to test our hypothesis with sufficient statistical power affordably.
Surgical model: RIC model involves a two-step surgery. In the first stage surgery, the tendon is fully detached at the enthesis and the animal recovers for 6 weeks. During this period of detachment the muscle tendon complex retracts, and, is also subjected to fatty infiltration as we reported earlier (53) (
We will compare reconstitution of enthesis under three groups: biphasic OsTend scaffold repair (OT), repair performed by monophasic scaffold (MO, pure collagen scaffold without HA deposition on distal end), and intact contralaterals (IC, positive control) will be the third group implicitly. There will be two time points of 2 weeks and 12 weeks. Previously we observed both 3 month (5) and 6 month (54) time points and healing strength was comparable; thus, 3-months is chosen as the end-point. We will operate on 12 rabbits/group/time point (N=48, equally distributed as male and female New Zealand White Rabbits). Scaffolds from 8 rabbits/group will be used for mechanical testing and the remaining four will be used to quantify healing immunohistologically.
In vivo assessment of joint healing biomechanics: Joint strength and joint range of motion will be measured as functional repair outcomes in vivo. Measurements will be performed longitudinally over time for each rabbit: 1) healthy reference baseline prior to the first surgery before induction of detachment, 2) immediately after first surgery after detachment to record detached baseline, 3) at 6 weeks at the end of the chronic degeneration period before the repair is performed as chronic baseline, 4) at 6 weeks after the repair surgery is performed, 5) after the repair surgery prior to the sacrifice. Infraspinatus muscle contractions will be induced using stimulation while the rabbit is already under anesthesia to assess shoulder strength and range of motion. Charge-balanced, cathodic stimulation will be delivered through stimulating electrodes (TECA Monopolar Needle Electrodes #902-DMG75-TP) inserted at the suprascapular notch by a custom-made programmable stimulator. To assess strength, the forelimb will be fixed in a stereotactic frame with the paw secured to a load cell while stimulation is delivered, as done in feline studies (55). Trains of 15 short tetanic bursts (1 second each separated by 5 seconds at 100 Hz, 5 mA 56) will be used to obtain mean force±standard deviations. Stimulus intensities that evoke 50% of the maximum force (as measured prior to induced degeneration during Surgery #1) will be used for comparison across time points to avoid reinjury of the healing tendon. ROM will be assessed using motion capture (Dual camera 3D Image system. Correlated Solutions Inc.) while the forelimb moves freely in response to both passive manual stretching and active contraction from the same stimulation protocol. Because each animal will be followed longitudinally, a paired-test will be used (Wilcoxon or paired t-test if normal) to assess convergence to healthy reference baseline, or improvement from detached/chronic baselines.
An experimental design for animal studies is provides in TABLE 1. This experimental design involves testing two time points, 2 weeks and 12 weeks, along with quantifying intact controls as the baseline.
H: Immuno/histological; M: Mechanical; * H for intact group is available from prior studies as embedded blocks; N=12/group/time point for joint mechanics and microCT.
Fatty infiltration: Scapulohumeral complexes will be dissected bilaterally and subjected to micro-CT (Siemens Inveon, 75-80 kV, 470-500 pa, 630-670 ms exposure, 21 μm voxel edge lengths). Gray level intensity will be calibrated by using phantom tissues. CT data will be processed (Matlab) to measure fatty infiltration in the muscle as we have done previously (53) (
Bone volume: Cortical thickness, trabecular thickness and BV/TV at the humeral head will be quantified using CT scans at two locations: a) repair site (i.e. the former trough that is filled with the distal segment of OsTend scaffold), b) lateral to the repair site to determine effects of repair remotely at the humerus.
Mechanical Testing: The length, width, and thickness of the regenerated tendon portion will be measured by custom-made electrical contact sensors. Bone-tendon attachment region will be 3D-scanned using a stereoimaging system (Correlated Solutions) from which footprint attachment area will be measured. Samples will be gripped at the muscle using a clamp cooled with liquid nitrogen. Humeral shaft will be potted in PMMA. Samples will then be preconditioned at 5 N amplitude cyclic load for 50 cycles, and loaded to failure (5 mm/sec). Load-displacement curves will be recorded to obtain the failure load and deformation. Stiffness will be calculated using linear regression. Failure strength and location will be recorded. Ultimate stress & strain and modulus will be calculated by using the apparent area depending on failure location (generally enthesis, sometimes mid-substance). Baseline repair strength will be measured by (N=8/group) implanting scaffolds in cadaveric rabbit shoulders using described surgical procedures and testing mechanically.
Quantitative Histology: Samples will be fixed in neutral buffered formalin and demineralized in EDTA for quantitative histomorphometry. Sections will be prepared for histology. Volume fractions of ELAC threads and de novo collagen deposition will be measured by point counting of Masson's trichrome stained sections where acellular ELAC threads can be easily distinguished from de novo fibrous tissue (5, 6). Picrosirius stained sections will be imaged under cross polarization (Olympus BX51) to obtain the fraction of fibers oriented parallel to the longer axis of the tendon as before (57). Immunohistology will be performed for Type I/III collagen, tenascin-C, tenomodulin, and decorin. Based on a systematic review of tendon repair scoring methods (58), Watkins method (59) is accepted as one of the more complete scoring systems. This method involves composite evaluation of scores for cellularity, cell morphology and collagen alignment to obtain a summative score wherein higher sums represent proximity to a tendon-like tissue.
Safranin-O stain will be used to label fibrocartilage. Histological scoring will be as defined by Yokoya et al. (51) which has been used by several other groups (28, 48). Scoring criteria are: continuity at insertion (C), alignment of fibrous tissue (R), presence of fibrocartilage (F), presence of tidemark (T). C+R−F−T− is scored as 1, C+R+F−T− is scored as 2, C+R+F+T− is scored as 3, and C+R+F+T+ is scored as 4. Using ImageJ software, total area of fibrocartilage phase will be traced. Picrosirius red sections will be imaged under circular polarization to measure the angular deviation of collagen fibers as a measure of overall orientation of de novo tissue (60). As illustrated in
Timelines and activities are provided in TABLE 2.
The surgical model is close to the previous tendon repair models that we published on with the exception that the trough in the bone is deeper by 4 mm (5, 54). We performed the surgery on one pilot rabbit which affirmed that deeper trough into the trabecular space did not affect the recovery and ambulation of the animal. Therefore, we do not anticipate problems in the surgical model.
We expect functional outcomes of repairs performed by using OsTend scaffolds to exceed those repairs that used monophasic scaffolds. We also expect the biphasic group to demonstrate greater evidence of an enthesis-like regeneration per section 1.2.2.2. Healing biology will involve the presence of a distinct fibrocartilaginous transition at the interface for the TC group as confirmed by immunohistological markers. We also expect the OsTend scaffold group to bear the greater load at smallest footprint among all groups, accordingly, stress bearing capacity of biphasic group will be superior.
If the emerging results from the first set of OsTend scaffold-treated rabbits show low-level of mineralization we may resort to seeding scaffolds with autologous mesenchymal stem cells that are collected at the time of the first surgery to induce chronic defect. We have experience in isolating and proliferating rabbit mesenchymal stem cells and their in vivo delivery with collagen scaffolds (5, 6). We think, though, that there is reasonable likelihood that simpler biomaterial only approach may work given the preliminary data (
In statistical analysis of a single variable, one-way ANOVA will be used (or Kruskal Wallis should Anderson-Darling normality test fails). Post hoc comparisons will be done in the last step using Tukey's post-hoc test (or Mann-Whitney with Bonferroni correction, if non-parametric). Significance will be set at p<0.05 for all studies after adjusting for multiple comparisons. Effect size: For Aim 2 that is testing our overarching hypothesis, the effect size is defined by success benchmarks: biomechanically attainment of material properties that are within 25% those of intact tendon (i.e. three quarters as strong as intact tendon) will define a treatment group that has attained an acceptable level of repair. At this effect size, a sample size of 8 for biomechanical strength, at 15% data scatter will exceed 80% power. The power will be greater than 80% for in vivo joint mechanics measures for which sample size will be N=12.
| Number | Date | Country | |
|---|---|---|---|
| 63488632 | Mar 2023 | US |
