Patent Application
20220001077
More than 500,000 surgical procedures are performed each year for damaged ligaments or tendons, and these connective tissue (CT) surgeries have long recovery times. CT injuries are of great concern to individuals with physically demanding professions, active lifestyles, or the elderly. CT injuries heal slowly and often incompletely, resulting in considerable pain, suffering and a decrease in quality of life. Quadriceps, Achilles, rotator cuff, and biceps tendons are the most common tendon injuries, with ruptures of the supraspinatus tendon in the rotator cuff affecting an estimated 22.1% of the population. Tendon rupture can occur from acute injury or through chronic degradation of the tissue in the absence of an apparent injury via an unknown biological mechanism. This lack of knowledge that underlies tendon breakdown results in limited and inadequate management options for tendon disorders. Clinical guidelines for a ruptured Achilles tendon recommend 6-12 weeks of immobilization and up to 4 months of recovery, while rotator cuff repairs are generally recalcitrant. Despite intense scientific focus, there has been little improvement in the rate or quality of tendon or ligament repair. A major advance in the repair of damaged CT would be a substantial reduction in healing time and an increase in the strength of repair.
In the United States alone, there are over 100 million injury-related medical appointments each year, with 60-67% specific to musculoskeletal injury. The direct healthcare expense for musculoskeletal conditions is approximately $130 billion annually. Many of the soft tissue musculoskeletal injuries involve tendons and ligaments and often require surgical intervention. Perhaps the most staggering statistic is that over 288 million workdays are lost annually due to musculoskeletal conditions, accounting for 73% of total work days lost. Compensation, lost wages, and lost productivity further burden the economy by approximately $50 billion every year. Workdays lost to slow or incomplete healing are a major issue. For example, the most common medical recommendation following tendon rupture is four months of recovery for those performing manual labor. Studies have shown that the mechanical properties of injured CT may actually take years to approach the properties of the uninjured state. The prevalence of rotator cuff injuries is another source of concern. Rotator cuff tears affect 40% or more of patients older than 60 years and are common causes of debilitating pain, reduced shoulder function, and muscle weakness. Repeat surgeries that are intended to address re-tears or re-ruptures are another area with significant potential for improvement. When repaired, rotator cuffs often form a recurrent tendon re-tear after surgery, often attributed to the patient's age or health, the size of the tear, or the level of tendon degeneration prior to surgery. Given the significant numbers of people negatively affected by tendon damage and the clear deficiencies in outcome, particularly with rotator cuff damage, new approaches should be explored.
Collagen is the predominant extracellular matrix molecule in load-bearing tissues. An important characteristic of collagen type I is its well-understood hierarchical structure from the nanoscale to the macroscale. Organizational motifs of collagen are very tissue specific, with the morphology reflecting the mechanical environment. The intricate and highly organized architecture of collagen materials leads to specific physical and biological functions that is characteristic across length scales. The self-assembly properties and biocompatibility of the collagen molecule make it a premier candidate for tissue engineering and regenerative medicine.
Current therapeutic approaches to damaged or diseased tendon involve implantation of rehydrated type I collagen gels, occasionally alongside the delivery of embedded stem cells or therapeutics. This collagenous network is sutured onto the damaged tendon where host cells infiltrate and remodel the implant. Since the implant is sutured directly onto the tendon, it also functions to increase the effective diameter of the tendon and therefore decrease the peak strain of the host tissue. This is hypothesized to protect the tendon from further tear progression as it undergoes remodeling. However, despite advances in fabrication and implantation techniques of these scaffolds, complete healing of collagenous tissues remains elusive, with only marginal increases in clinical scores and significant recurrence of wounds and tears. This lack of healing has been attributed to the paucity of native cell populations and their inability to mount a repair response. Reliance on scarce host cells to infiltrate and remodel these implants, even in the presence of numerous growth factors, is therefore insufficient.
The present technology provides compositions and methods for the controlled delivery of exogenous collagen to damaged connective tissue. The compositions and methods can be used to accelerate and improve wound repair by self-assembly of collagen. The compositions are provided in form of both stable and metastable liquid crystal collagen, which can form various networks or scaffolds. The stable and metastable liquid crystal collagen compositions can be formulated into injectable particles or gels. Injections of these materials can be used to treat various forms of connective tissue injury, or to prevent or slow degeneration secondary to cellular senescence for particularly vulnerable tendons that are generally refractory to repair when they fail (e.g. supraspinatus, flexor tendons, and Achilles tendon).
The present technology utilizes new materials to accelerate connective tissue repair by the controlled delivery of active collagen (i.e., tropocollagen monomer). Active collagen-mediated repair utilizes data and a theory, which show that collagen fibrillogenesis and stability are promoted by mechanical force. The concept that force directly drives collagen fiber formation (referred to as Mechanochemical Force Structure Causality or MFSC) suggests that if there is sufficient available active collagen (tropocollagen), and intermittent tensile mechanical forces are applied to damaged tissue, then repair fibrils are formed, damaged fibrils are repaired, and both are retained preferentially in the path of the load. The premise of MFSC has been clearly demonstrated in a cell-free system.
Collagen retention and assembly occur in the path of mechanical force. Retention is the effect whereby strain stabilizes collagen against thermal and enzymatic degradation. Mechanical strain can directly drive collagen assembly along force lines. Together, these two effects make it possible to provide sufficient active collagen to an injury or defect undergoing mechanical perturbation (e.g. ambulation, physical therapy, controlled passive loading, electromyography, ultrasound, vibration therapy, etc.) which then leads to faster injury repair and increased organization of the repaired tissue. It is important, however, to provide sufficient active collagen to the defect edges to facilitate recruitment and polymerization via flow-induced crystallization (FIC) or preferential incorporation of collagen molecules into damaged tissue.
An aspect of the present technology is the development of a highly-densified, liquid crystal collagen material with high stability (stable liquid crystal collagen, or SLCC), or a scaffold containing such material (stable liquid crystal collagen scaffold, or SLCCS). The inventors have generated collagenous sheets with controlled fibril alignment that persist over 7 orders of magnitude in length scale and which possess collagen densities that are about 3-4 times that of native tendon (>800 mg/ml).
Another aspect of the technology is the development of a highly-densified, metastable liquid crystal collagen (MLCC) material with tunable stability, or a scaffold containing such material (MLCCS). The material shown in
Both SLCC and MLCC can be introduced into a damaged connective tissue of a mammal, including a human, where they can act as organizational guidance structures to direct the repaid of aligned tissues, such as collagen-containing connective tissue. In particular, they can be used to aid in tendon repair by providing or guiding replacement of all or part of missing or damaged tissue.
Still another aspect of the invention is a bi-stable active collagen patch. A strong, bi-stable patch can be formed by fusing sheets of SLCC & MLCC collagen (
Yet another aspect of the technology is a process of creating SLCCS by a process termed “osmotic breathing”. For example, collagen can be dehydrated for 12 hours at 4° C. against acidic 10% polyethylene glycol dissolved in deionized water. The collagen solution can then be placed into a fresh dialysis bag which is placed in acidic 40% polyethylene glycol dissolved in deionized water. The collagen solutions can be subjected to ultrasound for 30 minutes, twice per day, over the course of 7 days. The dialysis bags can then be transferred to 40% polyethylene glycol dissolved in 1×PBS at 7.4 pH at 35° C. and then to 1×PBS solution at 35° C. at 24-hour intervals over the course of 7 days.
Another aspect of the technology is a process of creating MLCCS by osmotic breathing. The process can include first dehydrating a collagen solution by dialysis against acidic 10% polyethylene glycol dissolved in deionized water for 12 hours at 4° C. The solution is then placed into a fresh dialysis bag which is placed into acidic 40% polyethylene glycol dissolved in deionized water for 7 days at 4° C. The dialysis bag is then transferred to neutralized 40% polyethylene glycol dissolved in 1×PBS for 4 days at 4° C. The PEG/collagen solution is then warmed to 37° C. to initiate fibrillogenesis for 48 hours.
Still another aspect of the technology is a process of fabricating MLCCS via molecular crowding, in which liquid crystal phasing of collagen mesogens is developed through dialysis against, for example, acidic 40% polyethylene glycol (PEG). Once the mesogen concentration stabilizes (at about 300 mg/mL), “metastable fibrillogenesis” is induced via exposure to PEG/1×PBS at pH=7.4. In some embodiments, SLCCS can be produced by a similar process but with two procedural changes: 1) During the initial dialysis, the densifying collagen solution is subjected to two 30-minute treatments per day of, for example, 130 W ultrasound and 2) After 7 days, the sample is subjected to a 7-day PEG-PBS cyclic “osmotic breathing” treatment comprising alternating submersion into neutralized concentrated PEG-1×PBS and only 1×PBS at 37° C. at 24-hour intervals to induce “stable fibrillogenesis”.
Yet another aspect of the technology is a process of assembling a combined MLCC/SLCC bi-stable patch by polymerizing a dense (e.g., 15 mg/mL) solution of neutralized collagen between the MLCC and SLCC sheets and subjecting the patch to neutral 40% PEG-1×PBS at 37° C. for 24-hours. In some embodiments, UVA cross-linking can then be utilized, and SLCCs are cross-linked by exposure to UV and/or riboflavin per a modified Dresden protocol. An original cross-linking procedure, commonly referred to as the Dresden protocol, calls for UVA exposure for 30 minutes at 3 mW/cm−2. UV exposure time from 10 to 40 minutes is used to achieve a range of cross-linking densities as assessed by denaturation temperature. In some embodiments, for each class of material produced, sheets are cut such that long-axis is perpendicular to the birefringent texture (parallel to fibril orientation).
In a further process of the technology, the release rates of MLCC are tuned by altering the duration of ultrasound (US) and/or other conditions. In some embodiments, MLCC and SLCC are implanted or injected (after being fragmented) to serve as a tissue filler in a subject. In some embodiments, ultrasound is applied to the subject to convert the MLCC to SLCC and fuse the particles into a more solid, degradation-resistant state that possesses an overall lower energy.
The present invention is further summarized by the following list of items:
1. A non-naturally occurring, stable, liquid crystal collagen composition, wherein the composition comprises type I, II, III, V, and/or XI collagen organized into fibril-like structures 50-200 nm in diameter, wherein the fibril-like structures are locally aligned along their longitudinal axis and have D-periodic banding structure larger than about 67 nm, and wherein the composition has a density of at least about 500 mg/mL.
2. The collagen composition of 1 above, wherein the fibril-like structures shed monomeric collagen at a rate less than 1% per day at 37° C.
3. The collagen composition of 1 above, wherein the composition has a density of about 800 mg/m L.
4. A non-naturally occurring, metastable, liquid crystal collagen composition, wherein the composition comprises type I, II, III, V, and/or XI collagen organized into fibril-like structures 50-100 nm in diameter, wherein the fibrils are locally aligned along their longitudinal axis and substantially lack native D-periodic banding structure, and wherein the composition has a density of about 200-400 mg/mL.
5. A bi-stable liquid crystal collagen composition comprising the composition of 1 above and the composition of 4 above.
6. The bi-stable liquid crystal collagen composition of 5 above, wherein the relative amounts of the composition of 1 and the composition of 4 are selected to provide a desired collagen delivery rate in a collagen assembly process.
7. The composition of any one of 1-6 above configured as a plurality of particles, a gel, or a tissue scaffold.
8. The composition of 7 above configured as a tissue scaffold, wherein the scaffold further comprises one or more types of cells and/or one or more therapeutic agents.
9. The composition of 8 above, wherein the one or more therapeutic agents are selected from the group consisting of anti-inflammatory molecules, matrix anabolic molecules (signaling molecules which drive matrix assembly e.g. TGF-β3 and molecules directly involved in matrix construction e.g. fibronectin), matrix catabolic molecules (signaling molecules which drive matrix destruction e.g. IL-1 and molecules directly involved in matrix destruction e.g. matrix metalloproteases), proteoglycans, and glycosaminoglycans.
10. A method of making a stable liquid crystalline collagen composition, comprising the steps of: (a) concentrating a solution comprising type I, II, III, V, and/or XI collagen under acidic conditions; (b) subjecting the product of (a) to periodic sonication for several days; (c) osmotically cycling, neutralizing, and warming the product of (b) to form the stable liquid crystalline collagen composition.
11. The method of 10 above, wherein step (a) comprises dialyzing the collagen solution against an aqueous solution comprising about 10% polyethylene glycol having a molecular weight of about 35000 Daltons at a pH of about 1.5-2.5.
12. The method of 11 above, wherein step (a) comprises concentrating the collagen solution to about 10 to about 15 mg/mL.
13. The method of 11 or 12 above, further comprising the step of: (a1) dialyzing the product of step (a) against an aqueous solution comprising about 40% polyethylene glycol having a molecular weight of about 35000 Daltons at a pH of about 1.5-2.5 for about 7 days.
14. The method of any of 10-13 above, wherein step (a) is performed at about 4° C.
15. The method of 10 above, wherein step (b) comprises subjecting the concentrating collagen solution to sonication at 50-500 watts power, such as 130 watts power, and 5 kHz-50 kHz, such as 20 kHz, for 5-120 minutes from 1-5 times/day, such as 30 minutes twice per day.
16. The method of 10 above, wherein the osmotic cycling of step (c) comprises alternating dialysis of the concentrating collagen solution against (i) 40% polyethylene glycol in phosphate buffered saline at pH 7.4 and (ii) phosphate buffered saline at pH 7.4.
17. The method of any of 10-16 above, wherein warming of step (c) comprises performing said osmotic cycling and neutralization at about 35° C.
18. The method of any of 10-17 above, wherein the stable liquid crystalline collagen is stable under conditions of mechanical load, enzymatic cleavage, and solvent dissolution.
19. A method of making a metastable liquid crystalline collagen composition, comprising the steps of: (a) concentrating a solution comprising type I, II, III, V, and/or XI collagen under acidic conditions; (b) neutralizing and warming the product of (a) to form the metastable liquid crystalline collagen composition.
20. The method of 19 above, wherein step (a) comprises dialyzing the collagen solution against an aqueous solution comprising about 10% polyethylene glycol having a molecular weight of about 35000 Daltons at a pH of about 1.5-2.5.
21. The method of 20 above, wherein step (a) comprises concentrating the collagen solution to about 10 to about 15 mg/mL.
22. The method of 20 or 21 above, further comprising the step of: (a1) dialyzing the product of step (a) against an aqueous solution comprising about 40% polyethylene glycol having a molecular weight of about 35000 Daltons at a pH of about 1.5-2.5 for about 7 days.
23. The method of any of 19-22 above, wherein step (a) is performed at about 4° C.
24. The method of any of 19-23 above, wherein step (b) comprises dialysis of the concentrating collagen solution against 40% polyethylene glycol in phosphate buffered saline at pH 7.4.
25. The method of any of 19-24 above, wherein the warming of step (b) comprises performing said neutralization at about 35° C.
26. The method of any of 10-25 above, further comprising adding one or more anti-inflammatory molecules, matrix anabolic molecules, matrix catabolic molecules, proteoglycans, and/or glycosaminoglycans to the collagen solution at any step of the method.
27. The method of any of 10-26 above, further comprising applying tensile loading to the composition at any step of the method, optionally with the addition of monomeric type I, II, III, V, and/or XI collagen to the composition.
28. A method to aid in repairing damaged connective tissue in a human or other mammalian subject, the method comprising the steps of:
(a) providing a liquid crystalline collagen composition of any of claims 1-9; and
(b) placing said composition into a region comprising damaged connective tissue in the subject;
whereby said damaged connective tissue is at least partially repaired.
29. The method of 28, wherein step (a) comprises:
(a1) obtaining cells from the subject;
(a2) culturing the cells and optionally inducing differentiation and/or proliferation of the cells and/or collagen secretion by the cells;
(a3) harvesting collagen from the culture; and
(a4) forming a liquid crystalline collagen composition of any of claims 1-9 using the harvested collagen.
30. The method of 28 or 29, further comprising the step of:
(c) performing mechanical stimulatory therapy of said region.
31. The method of 30, wherein said mechanical stimulatory therapy comprises one or more of continuous passive motion, ultrasound, vibration, and electromyostimulation.
32. The method of any of 28-31, wherein step (b) is performed continuously or intermittently over a period of time.
33. The method of any of 28-32, wherein the liquid crystalline collagen composition further comprises one or more additional extracellular matrix molecular component.
34. The method of 33, wherein the additional extracellular matrix molecular component is a protein selected from the group consisting of other forms of fibrillar collagen, elastin, and fibronectin.
35. The method of 34, wherein the type I, II, III, V collagen, other form of fibrillar collagen, elastin, and/or fibronectin are obtained from a different species than the human or other mammalian subject.
36. The method of 34, wherein the type I, II, III, V collagen, other form of fibrillar collagen, elastin, and/or fibronectin are obtained from stem cells isolated from the subject.
37. The method of any of 28-36, wherein the damaged connective tissue comprises a wound, broken or fractured bone, ruptured tendon, hernia, damaged barrier membrane, or inflammation of a connective tissue.
38. The method of any of 28-37, wherein the method speeds repair of the damaged connective tissue.
39. A method of altering a chemical or physical property of the liquid crystalline collagen composition of any one of 1-9, the method comprising: (i) adding the composition to a scaffold; (ii) stacking rolling, or bending the composition; or (iii) mineralizing the composition with calcium phosphate.
40. A method of growing cells in culture, the method comprising adding the liquid crystalline collagen composition of any one of 1-9 to a cell culture, whereby cells in the culture become bound to or aligned with fibril-like structures in the composition.
41. A medical device comprising the liquid crystalline collagen composition of any one of 1-9
42. A tissue scaffold comprising the liquid crystalline collagen composition of any one of 1-9.
43. The tissue scaffold of 42, further comprising a plurality of cells, preferably stem cells, and more preferably mesenchymal stem cells.
44. The composition of 4, wherein the fibril-like structures further comprise
contrasting layered bands having a layer tilt angle of ˜45° compared to a longitudinal axis of the fibril-like structures, the bands comprising chevron-like defect structures and a chevron-like interface in a center of the structures.
Efforts to produce 3D fibrillar collagen scaffolds with biomimetic densities and organizational motifs for tissue engineering and regenerative medicine have previously resulted in limited success. Scaffolds obtained by previous methods often exhibit organizational motifs that persist over very small local distances or fail to maintain characteristic fibril morphologies of native tissue (80-200 nm diameter fibrils with ˜67 nm periodic banding). The approach of the present technology is to utilize a biomimetic crowding/confining mechanism to densify and organize the collagen substrate in concert with ultrasound application and fluid-cycling to control fibril morphological characteristics.
The low level of tropocollagen at the site of injury is a major limiting factor in the repair process. Assembly of collagen has been characterized in the literature. Collagens and their complementary associated extracellular matrix, ECM, molecules (i.e., proteoglycans, MMPs, fibronectin, etc.), are part of an autonomous structural system which has the capacity to self-assemble into load-bearing elements, spontaneously, in the path of force, precisely where they are needed. The assembly of tropocollagen into load-bearing tissues at regions of high loading is a fundamental property of modern metazoans. Mechanical strains are known to drive structure formation in other ECM biopolymers (e.g. fibronectin) and to promote crystallization in synthetic polymer systems. Recent studies have shown that collagen, in particular, will both form and retain structures (e.g. fibrils) directly in the path of tensile strains (see
The present technology provides the ability to use a collagenous implant to act as a delivery vehicle for active collagen monomer. As part of the regular wound healing cascade, damaged or diminished collagen fibrils in the wound site are repaired by de novo collagen monomer fabricated in the local cell population. However, due to the paucicellular nature of tendon, there is a significant lack of monomeric collagen production in the wound site. Damaged fibrils in the tissue are therefore slow to repair. The materials and methods of the present technology offer the ability to sustain delivery of collagen monomers over an extended period of time. Delivering supplemental collagen monomer into the wound in a controlled manner is thus a promising therapeutic approach.
The present technology provides a Stable Liquid Crystal Collagen Scaffold (SLCCS) cohesive network of collagen fibrils organized into a chiral smectic liquid crystal array (and with potential for other LCC organizational motifs). The fibrils are banded and aligned, and the fibril arrays change orientation by +/−70 degrees at ˜20 micron intervals; these parameters can vary by +/−5%, 10, or 20%. The collagen fibril network is produced via inducing liquid crystal phasing of collagen and dehydrating before condensation of monomers into densely-packed fibrils. The fibril network contains pores which can be used to incorporate therapeutics/cell lines. The technology provides tunable fibril and pore characteristics via promoting/preventing nucleation and growth phases of fibril precipitation. Altering these characteristics can alter delivery rate of monomer, porosity, and scaffold mechanical properties.
Another aspect of the present technology is an SLCCS substrate or scaffold containing aligned, banded collagen fibrils whose orientation persists over the scale of centimeters. The fabrication of the substrate is easily scaled-up and can be used to build large substrates or tissue equivalents by stacking SLCCS sheets. Nanoscaled and microscaled pores present in the substrates are orders of magnitude smaller than those found in dehydrated collagen gels of the prior art, and their size can inhibit disadvantageous burst release of therapeutics contained in the pores.
The SLCCS collagen network and scaffold of the present technology offer tunable fibril characteristics and density, which permits the formulation of constructs that mimic particular native collagenous tissues. The scaffolds can be stacked or rolled to create collagenous tissue equivalents (both with and without cells). The collagen networks and scaffolds find application in cornea, bone, tendon, and ligament tissue engineering, and can be seeded with therapeutics or stem cells for applications in wound healing. They also can be used for in vitro laboratory studies, such as investigations of the effect of aligned collagen on stem cell behavior and the effect of collagen fibril alignment on the rate of drug delivery.
SLCCS scaffolds consist of >95% collagen type I fibrils condensed into a dense fibrillar sheet. SLCCSs are fabricated via inducing liquid crystal phasing of a dense collagen solution prior to initiating fibrillogenesis. The organizational motif resembles a chiral smectic liquid crystal, with fibrils aligned along their long-axis into fibril arrays. At ˜20 micron intervals, the orientation of fibril arrays reorients ˜70 degrees and returns to the baseline orientation after an additional 20 microns; these parameters can vary by +/−5%, 10, or 20%. This morphology resembles a chiral smectic liquid crystal and to our knowledge, collagen scaffolds with this organizational motif have never been reported in the literature. Most importantly, this organizational pattern persists uninterrupted over a length of at least 45 millimeters and represents a significant advancement in the development of collagen scaffolds with long-range order. Fibrils within the SLCCS recapitulate the nano- and micro-hierarchical organizations of native collagenous tissues, while maintaining characteristic morphologies of native fibrils. While the organization of SLCCS span over a considerable distance, the macroscale organization resembles a chiral smectic liquid crystal and does not recapitulate the macroscopic organization of native tissues.
SLCCSs have potential applications in a number of tissue engineering and regenerative medicine applications. SLCCS can be fabricated as a thin sheet of predictably organized collagen. These sheets can be used for laboratory research for investigators examining the effect of aligned collagen on cell behavior and diffusion of small molecules in anisotropic matrices. SLCCS sheets also can be stacked to create larger 3-dimensional structures resembling cornea, bone, tendon, and ligament for tissue engineering applications. Furthermore, SLCCS can serve as a scaffold for cell and therapeutic delivery in regenerative medicine.
Characteristics of SLCCS collagen networks and scaffolds, including collagen concentration, dimensions, fibril morphology, and pore size, can be adjusted via manipulating the parameters of the liquid crystal system, including length of time dehydrating in polyethylene glycol, presence of ionic species, concentration of ionic species, temperature, length of time in temperature, confining geometries, and application of mechanical stimulation. Further modifications to the SLCCS include addition of exogenous crosslinking, proteoglycans, non-collagenous proteins, glycosaminoglycans, and other fibrillar collagens (II,III,V,XI) to control mechanical and morphological properties of the scaffold.
Another aspect of the SLCC materials is the mechanical anisotropy.
As demonstrated in
The present technology further provides a fabrication technique for producing Metastable Liquid Crystal Collagen Scaffolds (MLCCS) for the purpose of rapid delivery of large quantities of collagen monomer to collagenous defects. These scaffolds consist of >95% collagen type I fibril-like structures condensed into a dense fibrillar sheet. Most notably, the collagen fibrils exhibit a unique morphology, which is characterized by collagen fibrils that lack the characteristic D-periodicity of native fibrils.
The MLCCS contains metastable liquid crystal collagen (MLCC) material with tunable stability.
This periodic banding pattern of a fibril is a distinguishing feature of fibrils and arises from the precise lateral arrangement of molecules within a fibril. A lack of D-periodicity suggests that monomers residing in the MLCCS fibrils are not situated into proper positional configurations and are therefore more susceptible to dissociation from the fibrillar scaffold. Once dissociated, monomer will self-assemble into new and existing fibrils of the host tissue thus accelerating wound repair. MLCCSs are extremely dense and have the potential to deliver monomeric collagen to a wound site over a period of weeks to months.
The present approach is a departure from current therapeutic strategies for the repair of collagenous defects. MLCCS compositions can be used as a standalone implant or in conjunction with other available collagen products. A scaffold containing MLCCS features tunable delivery rates of collagen monomer to make it suitable for a range of defects and therapies. The scaffold also features nanometer- and/or micrometer-size (diameter) pores capable of transporting additional therapeutics or human cell lines.
MLCCS collagen networks are cohesive fibrous networks of aligned collagen fibrils which lack characteristic 67 nm D-periodicity. The fibrils exist in arrays that exhibit undulating morphology instead of typical arrays of parallel, linear fibrils. The high density undulating fibrils leave pores in network for therapeutics. The fibril networks are produced via inducing liquid crystal phasing of collagen, with dehydrating before condensation of monomers into fibrils. The fibril and pore characteristics are tunable via promoting or preventing nucleation and growth phases of fibril precipitation. Altering these characteristics alters delivery rate of monomer, porosity, and scaffold mechanical properties. The stability of MLCCS can be tuned through exposure to ultrasound, thermal cycling or crosslinking agents.
Another aspect of this material is the tunable mechanical properties as a function of applied ultrasound durations. Modulating the duration of ultrasound during the dehydration phase of assembly from 5, 10, and 30 minutes results in an increase in the mechanical integrity of collagen scaffolds. In all cases, tangent modulus significantly increases relative to MLCC materials (mean 0.73±0.02 MPa) with applied ultrasound as shown in
MLCC collagen fibrils have a higher free energy than dehydrated collagen gels used in current therapeutics and can deliver monomer at a faster rate. An MLCCS construct has a significantly higher density and lower stability than dehydrated collagen gels used in current market products. MLCC collagen networks and scaffolds thus contain a larger reservoir of collagen monomer that makes them ideal for delivering collagen monomer to host tissue over a period of time up to months. Pores of MLCC compositions are orders of magnitude smaller than dehydrated collagen gels of the prior art and will inhibit disadvantageous burst release of therapeutics. Their tunable pore size and density permits formulations of constructs for different therapeutics and/or cell types. Their tunable fibril characteristics and density permits formulations containing constructs that are tissue-specific and have different release characteristics.
Monomers can dissociate from the MLCCS fibrous network at a higher rate than the relatively stable monomers in previously available collagenous therapeutics containing native-like banded collagen. Once solubilized, monomer settles in its lowest free energy state and thus accumulates in the loaded native fibrils of the wound site, resulting in decreased time to healing. Monomers can also assemble along the path of the tissue strain to create de novo fibers.
Characteristics of the scaffold including concentration, fibril morphology, and pore size can be adjusted to alter delivery rate and profile of both collagen and additional therapeutics. This can be achieved via manipulating the parameters of the liquid crystal system, including length of time dehydrating in polyethylene glycol, presence of ionic species, concentration of ionic species, temperature, length of time in temperature, confining geometries, osmotic cycling, and application of mechanical stimulation. Potential modifications to the MLCC include addition of exogenous crosslinking, proteoglycans, glycosaminoglycans, non-collagenous proteins, and other fibrillar collagens (II,III,V,XI) to control mechanical and morphological properties of the scaffold.
Both SLCCS and MLCCS compositions can be used to treat injuries to tendon, ligament, and joint capsules, for example. Tendon injuries that can be treated include, for example, shoulder rotator cuff tendons, Achilles tendon, and patellar tendon.
Methods of producing SLCCS and MLCCS are further described below.
In one aspect of the technology, SLCCS can be created by a process termed “osmotic breathing”. In an embodiment of the process, a collagen solution is dehydrated by dialysis for 12 hours at 4° C. against acidic, with a pH of about 1.5 to 2.5, 10% polyethylene glycol (MW˜35000 Daltons) dissolved in deionized water. In some embodiments, the collagen solution is concentrated to about 10-15 mg/mL.
The collagen solution is then placed into a fresh dialysis bag which is placed in acidic 40% polyethylene glycol dissolved in deionized water. The collagen solutions are subjected to ultrasound for 30 minutes, twice per day, over the course of 7 days. The dialysis bags are then transferred to 40% polyethylene glycol dissolved in 1×PBS at 7.4 pH at 35° C. and then to 1×PBS solution at 35° C. at 24-hour intervals over the course of 7 days. In some embodiments, the sonication is at about 130 watts power and about 20 kHz. In some embodiments, ultrasound is necessary to stabilize tropocollagen into natively banded fibrils.
Another aspect of the technology is a process of creating MLCCS by osmotic breathing. An embodiment of the process includes first dehydrating a collagen solution by dialysis against acidic (pH about 1.5-2.5) 10% polyethylene glycol (MW˜35000 Daltons) dissolved in deionized water for 12 hours at 4° C. The solution is then placed into a fresh dialysis bag which is placed into acidic 40% polyethylene glycol dissolved in deionized water for 7 days at 4° C. The dialysis bag is then transferred to neutralized 40% polyethylene glycol dissolved in 1×PBS for 4 days at 4° C. The PEG/collagen solution is then warmed to 37° C. to initiate fibrillogenesis for 48 hours.
Yet another aspect of the technology is a process of assembling a combined MLCC/SLCC bi-stable patch by polymerizing a dense (e.g., 15 mg/mL) solution of neutralized collagen between the MLCC and SLCC sheets and subjecting the patch to neutral 40% PEG-1×PBS at 37° C. for 24-hours. Optionally, cross-linking can then be utilized (such as by UVA or another known method), and SLCCs are cross-linked by exposure to UV and/or riboflavin per a modified Dresden protocol. An original cross-linking procedure, commonly referred to as the Dresden protocol, calls for UVA exposure for 30 minutes at 3 mW/cm−2. UV exposure time from 10 to 40 minutes is used to achieve a range of cross-linking densities as assessed by denaturation temperature. In some embodiments, for each class of material produced, sheets are cut such that long-axis is perpendicular to the birefringent texture (parallel to fibril orientation).
In a further process of the technology, the release rates of MLCC are tuned by altering the duration of ultrasound (US) and/or other conditions. In some embodiments, MLCC and SLCC are implanted or injected (after being fragmented) to serve as a tissue filler in a subject. In some embodiments, ultrasound is applied to the subject to convert the MLCC to SLCC and fuse the particles into a more solid, degradation-resistant state that possesses an overall lower energy.
In some embodiments, MLCCS contain metastable liquid crystal collagen (MLCC) material with tunable stability. As shown in
Any of the materials of the present technology can be employed in methods of promoting or accelerating healing of damaged connective tissue. The damaged connective tissue can include a wound, such as a wound caused by trauma or infection, a broken or fractured bone, a ruptured tendon, a hernia, or inflammation in a connective tissue. The healing or repair can be undertaken by surgical implantation or injection of the material at or near the damaged site. The healing or repair also can include surgical, biochemical, pharmacological, genetic, physical, stem cell, or other treatment of the damaged connective tissue and/or other parts of the subject's body. Physical methods of treatment can include one or more of continuous passive motion, ultrasound, vibration, and electromyostimulation. The material can be provided as SLCCS, MLCCS, or a bistable patch containing SLCC and MLCC. The collagen in the material can be type I, II, III, or V collagen, or another form of fibrillar collagen. The collagen can be isolated from cells of the subject, isolated collagen from the same species as the subject, or isolated collagen from a different species. The material can contain one or more additional extracellular matrix molecular components, such as elastin or fibronectin, from the cells of the subject, from the same species as the subject, or from a different species.
Methods to aid in repairing damaged connective tissue as described above can accelerate healing of a damaged connective tissue, or reduce the time to full load bearing capacity after damage to connective tissue, or increase the stiffness or load bearing of a connective tissue at a given time after an injury. Such methods can be implemented in a human or other mammalian subject. The methods can include the following steps: (a) providing any of the liquid crystalline collagen compositions described herein; and (b) placing the composition into a region that contains damaged connective tissue in the subject. Following step (b), the damaged connective tissue is at least partially repaired or healed, or stiffness, or spring constant, or load bearing capacity of the connective tissue is increased. Step (a) of the method can include the following: (a1) obtaining cells from the subject; (a2) culturing the cells and optionally inducing differentiation and/or proliferation of the cells and/or collagen secretion by the cells; (a3) harvesting collagen from the culture; and (a4) forming any of the liquid crystalline collagen compositions described herein using the harvested collagen. Step (b) can be performed continuously or intermittently over a period of time.
Stable Liquid Crystal Collagen Scaffolds (SLCCS) were created by dehydrating 10 mL of telopeptide intact bovine collagen (TELOCOL, Advanced BioMatrix) against acidic 10% polyethylene glycol dissolved in deionized water (35,000 MW) for 12 hours at 4° C. The solution was extracted and placed in a fresh 45 mm×15 mm dialysis bag (3500MWCO) and then placed in acidic 40% polyethylene glycol dissolved in deionized water. These samples were subjected to ultrasound using a 3510 Branson sonicator for 30 minutes, twice per day over the course of 7 days. Samples were then transferred to 40% polyethylene glycol dissolved in 1×PBS at 7.4 pH at 35° C. and then to 1×PBS solution at 35° C. at 24-hour intervals over the course of 7 days. This process is termed “osmotic breathing”. The resulting scaffold contained 31 mg of type I collagen with dimensions of 45 mm×15 mm×50 microns and featured a fingerprint pattern under polarized light that is characteristic of liquid crystalline order. This fingerprint pattern persisted over >90% of the scaffold and corresponds to shifts in the alignment of fibril arrays (resembling a chiral smectic liquid crystal) in the X-Y plane.
The density of this material was about 800 mg/cm3. Fibrils were 80-100 microns in diameter and organized into aligned fibril arrays aligned along the long-axis of fibril. Fibrils had an ˜85 nm periodic banding structure that closely resembles, but does not duplicate, the D-periodicity of native collagen fibrils. This suggests that monomer in reconstituted fibrils were settled in stable 3-dimensional configurations.
Metastable Liquid Crystal Collagen Scaffolds (MLCCS) were created by dehydrating 10 mL of telopeptide intact bovine collagen (TELOCOL, Advanced BioMatrix) against acidic 10% polyethylene glycol dissolved in deionized water (35,000 MW) for 12 hours at 4° C. The solution was extracted and placed in a fresh 45 mm×15 mm dialysis bag (3500 MWCO) and then placed in acidic 40% polyethylene glycol dissolved in deionized water for 7 days at 4° C. The dialysis bag was then transferred to neutralized 40% polyethylene glycol dissolved in 1×PBS for 4 days at 4° C. The PEG/collagen solution was then warmed to 37° C. to initiate fibrillogenesis for 48 hours. The resulting scaffold contained 30 mg of type I collagen with dimensions of 45 mm×15 mm×100 microns and featured a fingerprint pattern under polarized light that is characteristic of liquid crystalline order. This fingerprint pattern persisted over >90% of the scaffold and corresponds to shifts in the alignment of fibril arrays in the X-Y plane.
The density of this material was about 200-500 mg/cm3. Fibrils were 80-100 microns in diameter and organized into undulating fibril arrays aligned along the long-axis of fibril. They also lacked the characteristic D-periodicity banding of native fibrils. This morphology is atypical in native tissues and suggests that monomers in reconstituted fibrils are settled in improper 3-dimensional configurations of a local energy minimum that is less stable than the SLCCS.
MLCC material were cross-sectioned, and the cut MLCC material was embedded in transmission electron microscopy resin. Surprisingly in
SLCC and MLCC materials were prepared as described in the above examples and compared. Mechanical specimens were prepared by cutting sheets are cut into 10 mm (5 mm max and 3 mm gauge width) dog-bone tensile test specimens. The strips were cut such that long-axis is perpendicular to the birefringent texture (parallel to fibril orientation).
Mechanical testing was performed within 6 hours post-fabrication, specimens were mounted between spring-loaded grips and preloaded to 0.002N in a custom mechano-bioreactor. All specimens were strained to failure at a rate of 6 mm/min while recording the load. Linear modulus (Pa), onset of plastic deformation (mm), ultimate tensile stress (Pa), and toughness (J) were determined.
As demonstrated in
Ultrasound duration was then varied and tested. MLCC materials received no ultrasonic treatment. SLCCS, SLCC10, and SLCC30 received 5, 10, and 30 minutes of ultrasound, twice per day during the dehydration phase of the fabrication protocol. In
Modulating the duration of ultrasound during the dehydration phase of assembly from 5, 10, and 30 minutes resulted in an increase in the mechanical integrity of collagen scaffolds. In all cases, tangent modulus significantly increased relative to MLCC materials (mean 0.73±0.02 MPa) with applied ultrasound as shown in
Type I telocollagen is acetic acid extracted from young (8-12 wks) New Zealand white rabbit scleras (Pel-Freez Biologicals, Rogers, Ark.).
MLCCSs (as in
SLCCSs (as in
Combined MLCC/SLCC bi-stable patch is assembled by polymerizing a dense (15 mg/mL) solution of neutralized collagen between the MLCC and SLCC sheets and subjecting the patch to neutral 40% PEG-1×PBS at 37° C. for 24-hours.
Modulating SLCC mechanics through UVA cross-linking: SLCCs are cross-linked by exposure to UV and riboflavin per a modified Dresden protocol. An original cross-linking procedure, commonly referred to as the Dresden protocol, calls for UVA exposure for 30 minutes at 3 mW/cm−2. UV exposure time from 10 to 40 minutes is used to achieve a range of cross-linking densities as assessed by denaturation temperature.
Mechanical specimens: For each class of material produced, sheets are cut into 10 mm (5 mm max and 3 mm gauge width) dog-bone tensile test specimens. The strips are cut such that long-axis is perpendicular to the birefringent texture (parallel to fibril orientation).
Mechanical testing: Within 6 hours post-fabrication, specimens are mounted between spring-loaded grips and preloaded to 0.002N a custom mechano-bioreactor. All specimens are strained to failure at a rate of 6 mm/min while recording the load. Linear modulus (Pa), onset of plastic deformation (mm), ultimate tensile stress (Pa), and toughness (J) are determined.
Release rates of the SLCC, the MLCC and combined SLCC/MLCC bi-stable patches is determined by the following procedure: Monomeric collagen is labeled using standard methods for fluorescently labeling antibodies that result in an average of two fluorophores per tropocollagen. Labeled monomer is added to unlabeled monomer in a ratio of 1:1000 and then incorporated into each collagen sheet.
Determining kinetics of active collagen (AC) delivery: Each class of material is fabricated as described previously. Five samples are cut from each collagenous sheet (collagen content ˜7.75 mg) and submerged in physiologic salt buffer at 37° C. Release solution is collected and replaced at 24-hour intervals.
Tuning kinetics of MLCC-AC delivery: To tune the release rates of MLCCs, the duration of ultrasound (US) is altered. US is necessary to stabilize tropocollagen into natively banded fibrils. MLCCS samples are fabricated with fluorophore-labeled monomers as described above. Experimental conditions include a 1, 5, 10, 15, 20, or 25-minute treatment of 130 W US twice per day for seven days over the course of dehydration against acidic PEG. MLCC sheets are divided into 5 mm×5 mm squares. Release solution is collected and replaced at 24-hour intervals.
Quantifying AC release rates: Aliquots of release solution are examined via fluorescence imaging using a Nikon inverted microscope (ECLIPSE TE2000-E) equipped with a high speed EMCCD Camera (iXon Ultra 897, ANDOR). Fluorescence intensity of a solution is converted to collagen concentration by use of a standard relating fluorescence intensity to collagen concentration. After 14 days, all samples are dissolved in 0.5M HCl to determine the amount of undelivered tropocollagen. Fractional delivery (FDcoll) as a function of time is determined.
Delivery of AC collagen produces a repair effect via MFSC on a simplified damaged tissue model and an excised, damaged native connective tissue in vitro. To demonstrate this process, collagen is delivered from a bi-stable collagen patch directly to models of damaged tissue that are subjected to mechanical stimulation. MFSC is demonstrated by participation of collagen from the patch in the repair of the defects in the tissue models. Two tissue models are used: 1) a dense disorganized collagen strip (DDCS), and 2) native rat tail tendon (RTT).
Collagenase tendinopathy model: To simulate tendinopathy, the tissue models are exposed to purified bacterial collagenase. Exposure time is standardized by tracking loss of constituent tropocollagen and measuring the change in tangent modulus.
Partial transection model: To simulate a focal defect, tissue models are partially transected to produce a central linear defect of 50% of the width of the strip (
Repair procedure: Bi-stable patches containing dense AC fabricated with labeled monomers (tropocollagen) are placed on the DDCS strip (or wrapped around the RTT), with the MLCC in direct contact with the “damaged tissue”. Specimens are mounted in a mechanobioreactor. Tensile strain is applied as a linear ramp at strain rates of 0.1, 1 and 10/s to 8% engineering strain, under the linear elastic limit of DDCS and RTT. Zero-strain and 8% static strain serve as controls for loading and for dynamic loading. The strain rate values are based on muscle contraction velocities and extensional strain rates required to induce collagen assembly. Strain is held at 8% for 5 minutes to permit ample time for re-equilibration of the collagen monomers with the sample and for viscous relaxation.
Quantitative determination of AC transfer: Tendon models are assessed via fluorescence imaging to quantify amount of fluorescently labeled collagen transferred to the repair site. Results are expressed as area labeled collagen (normalized for labelled collagen ratio)/area total collagen. In both partial transection models, the extensional strain rate varies from the center of the defect to the edges of the sample and along the sample's long axis, allowing comparison of the effect of varying extensional strain rates on collagen incorporation.
Mechanical testing: Following fluorescence imaging, samples are subjected to the mechanical testing protocol described above. Mechanical properties of repair outcomes are compared against mechanical properties of damaged tissue models.
The in vivo safety of MLCCS, SLCCS, and the bi-stable MLCC/SLCC rotator cuff patch is tested in a rabbit model. Immune and inflammatory responses in the tendon and local tissues are assessed, as well as any gross tissue changes.
Optimization of the patch configuration, location and fixation. Twelve shoulders from six New Zealand White (NZW) rabbit carcasses are used to test the patch position, fixation and retention on the supraspinatus tendon by articulating the joint mechanically for 12 hours while immersed in PBS in a simplified custom version of the Admet Mechanobioreactor.
NZW rabbits undergo bilateral shoulder surgery to assess the local cellular, neovascular and immune response of three collagen releasing patches at days 7, 14 and 28 on the subscapularis tendon and the surrounding tissues. Sham surgeries on contralateral shoulders serve as control. At the time of harvest the supraspinatus tendon and surrounding tissues are photographed to document the gross tissue response. Retrieved tendons are harvested, fixed in 10% NBF at 4° C., dehydrated and processed by standard paraffin embedding. Blocks with tendons are prepared for longitudinal and cross section sectioning to study the tendon, patch and their interface. Three 5 μm sections are obtained from each block and for longitudinal sections, three 5 μm sections are obtained at 2 tendon depths (sub-surface and mid-depth) and stained with H&E, Masson's trichrome and Picrosirius red. Adjacent tissues are also harvested and analyzed.
The punch model of Achilles tendon repair in rats was used to test the effectiveness of SLCC/MLCC bistable collagen patches in promoting tendon repair. A cylindrical defect was created in rat Achilles tendon using a punch, and treatment was induced in a group of the animals by placing a bistable SLCC/MLCC collagen implant within about 0.5-2 mm of the defect. The implant was aligned with the direction of collagen fibers in the tendon. After 14 days, tendons were removed from a treated group and an untreated group and their mechanical properties analyzed. The results shown in
MLCC and SLCC are implanted or injected (after being fragmented) to serve as a tissue filler in a subject. Following injection, ultrasound is applied to the subject to convert the MLCC to SLCC and fuse the particles into a more solid, degradation-resistant state that possesses an overall lower energy.
General Physiology 43, 265-283 (1959).
This invention was made with government support under Grant No. 1309579 awarded by the National Science Foundation. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/061329 | 11/13/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62760903 | Nov 2018 | US | |
| 62800945 | Feb 2019 | US |
