Patent Application
20210308333
The disclosure relates to compositions and uses of a material with a complex surface.
Each year, millions of metallic surgical implants are placed in patients worldwide, which include total hip replacements, dental implants and knee prostheses, screws to secure spinal fixation devices and anchorage components for facial prostheses, hearing aids and orthodontic appliances. With the exception of cemented prostheses, osseointegration is crucial to the functional success of such endosseous devices. Osseointegration may be achieved by either contact or distance osteogenesis—the formation of bone directly on the implant surface, or the old bone surface, respectively. While initial implant stability may be achieved by physical engagement in cortical bone, contact osteogenesis will only occur through bone remodeling. On the contrary, in the trabecular bony compartment, contact osteogenesis can provide rapid bony anchorage due to the recruitment and migration of osteogenic cells (osteoconduction) from the marrow interstices to the implant surface1.
Nanosurfaces have improved clinical osseointegration by increasing bone/implant contact. Neovascularization is considered an essential prerequisite to osteogenesis, but no previous reports have examined the effect of surface topography on the spatiotemporal pattern of neovascularization during peri-implant healing.
As well, improved compositions and devices are needed that promote neovascularization during wound healing.
Compositions, devices, methods, and uses are provided that promote neovascularization during wound healing, and are preferably based on improved osseointegration seen in some bone implants.
Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the disclosure are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.
An embodiment of the present disclosure will now be described in relation to the drawings in which:
Supplementary
Supplementary Movie Still 1 shows intravital live video of peri-implant neovasculature. Peripheral and central vasculature anastomosed on the top flat surface of the implant, facilitating the blood flow in each direction.
Supplementary Movie Still 2 shows μCT images of the entire healing volume around the NT Ti implant at day 42 post-surgery. The stack of the images along the Z axis shows different slices from the top (cover glass as seen in the ground glass appearance due to shadowing) through the depth of the healing volume. Formation of the bone on the surface of the implant is apparent. As the endocranial periosteum (dura mater) is approached, we see no signs of bone formation originating from the dural surface.
It is generally accepted that the mesenchymal progenitors of osteogenic cells are perivascular cells2,3, although little is known about how and when these cells enter the wound site. Neovascularization, or formation of new blood vessels, is a critical prelude to osteogenesis. Neovascularization may occur through either angiogenesis and/or vasculogenesis4,5; and it can be assumed that the incursion of perivascular cells is dependent upon neovascularization. Neovascularization may occur through a variety of mechanisms6-11 that lead, through maturation, to the establishment of a hierarchical functional vascular network. While implant surface design is considered a critical driver of osteoconduction, and topographically complex implants have been shown to increase bone-implant contact (BIC)12-14, no evidence has emerged to suggest that implant topography has an influence on peri-implant neovascularization.
It has been shown that implant surfaces increase platelet and neutrophil adhesion and activation15-17 that lead to an increased level of local angiogenic and osteogenic growth factors and cytokines18. Furthermore, micron-scale roughness on titanium (Ti) implants has been shown to stimulate the secretion of pro-inflammatory cytokines by macrophages including tumor necrosis factor (TNF)-α19, which primes endothelial cells for angiogenic sprouting20. Indeed, some authors have reported that rough implant surfaces affect endothelial cell proliferation, motility21, and endothelialization (tube formation)22. To complement these in vitro reports, upregulation of angiogenic and osteogenic genes has been reported following clinical insertion of topographically complex titanium implants23.
To determine the effect of surface topography on peri-implant healing, the inventors have developed a new in vivo experimental murine model to track the spatiotemporal development of neovascularization in the peri-implant healing compartment as a function of implant surface topography. The model integrates a custom-designed cranial metallic implant with an optically-transparent window chamber that is compatible with both confocal- and multiphoton-based intravital microscopic imaging systems.
From these models and studies, implant surfaces that promote osseointegration are utilized to develop materials that promote or enhance neovascularization for wound healing, such as subcutaneous or internal wounds.
In some embodiments, a composition for the promotion neovascularization during wound healing has a topographical complex surface, such as a micro- and/or nano-topographical complex surface. In one embodiment, the composition is made of a biocompatible material. Examples of biocompatible materials include, but not limited to: degradable synthetic and biological polymers, co-polymers, polymer blends, rubber, latex, silicone, carbon materials and inorganic materials such as metals, silicon, glass, ceramics and composite/alloy materials.
As used herein, a “topographical complex surface” means a surface structure in the micro or nano scale. In some embodiments, a topographical complex surface is comprised of microtubules, threading, pores, porous sinters, and/or microtextures. Examples of topographical complex surfaces are found in Koshy, E., and Philip, S. R. (2015); Smeets, R. et al (2016), and Stanford, C. M. (2010), the entire disclosures of which are incorporated herein by reference.
In some embodiments, the composition having micro- and nano-topographical complex surface can be formed into a product or device. In other embodiments, a product or device is provided comprising the composition having micro- and nano-topographical complex surface. Examples of such a product or device include, but are not limited to: skin dressing, bandage, scaffold, patch, implant, thin film, wire, catheter (insertion lines), meshes, nanowires, and implantable vascular beds.
Embodiments of the compositions can be of any size or shape or thickness and can be formed into in at least one of the product or device, or combinations thereof.
Compositions having a micro- and nano-topographical complex surface, and products or devised formed from such compositions can be used for modifying the rate, extent, location and directionality of vascularization (as well as cell migration and cytokine release) for tissue regeneration, cell therapy, organ transplantation, wound and defect healing, and cosmetic and agricultural engraftment applications. In one embodiment, the compositions, products or devices are used for modifying or enhancing the rate, extent, location and directionality of neovascularization during wound healing.
In some embodiments, the compositions, products, or devices are used in combination with biological components. In other embodiments, the compositions, products, or devices further comprise biological components. Examples of biological components include, but not limited to: tissues, cells, exosomes, extracellular vesicles, microparticles, cytokines, drugs, antibiotics, antifungal, anti-inflammatory, nanoparticles, and media.
In some embodiments, the compositions, products, or devices are used in combination with contrast agents. In other embodiments, the compositions, products, or devices further comprise contrast agents. Examples of contrast agents include, but are not limited to: fluorescent dyes, chromogenic dyes, quantum dots (QDots), Raman-active agents, molecular beacons, nanoparticles having fluorescent agents, and scattering or absorbing nanoparticles, biologically-activated/sensitive contrast agents (enzyme-cleavage, pH-sensitive, ROS-sensitive).
In some embodiments, contrast agents are used to label various components of the micro- and nano-topographical complex surface of the composition and any additional components, such as biological components. For example, the nanosurface could be optically labeled with a fluorescent dye of a specific fluorescence wavelength and impregnated cells could be labeled with another fluorescent dye of a different fluorescence wavelength, and each dye excited by different wavelength light sources. In this fluorescence multiplexed manner, different components of the embodiment can be labeled and tracked in a target over time to determine changes therein.
In tissue regeneration, cell therapy, organ transplantation, wound healing and cosmetic applications, the compositions, products, and devices are used to increase the loco-regional amount of functional blood vessels (as well as cell migration through and cytokine release from) a target tissue or organ or wound to improve the treatment thereof.
In some embodiments, the compositions, products, or devices are applied or inserted or administered or implanted in a target. As used herein, examples of a target includes, but are not limited to: a surgical field, a wound, a burn, a tumor, an organ, a tissue or cartilage or tendon, a scar target, a skin target, a biological target, a non-biological target, an oral target, an ear-nose-throat target, an ocular target, a genital target, a bladder target, a gastrointestinal target, a facial target, a cardiac target, a lung target, a bone and non-bone orthopedic target, a cartilage or spinal cord target, an anal target and a body target, a body defect target, a nerve target, a surgical cavity target, an engineered tissue construct, a plant material target.
In some embodiments, the products or devices described herein are used in ameliorating the adverse effects of aging or impaired healing conditions brought about by diseases impeding healthy functional vascularization e.g. diabetes, macular degeneration, or to increase or restore vascularization in skin grafts from autologous or substitute graft sources, increasing vascularity in damaged heart disease.
In one embodiment, the products or devices described herein employ one or more integrated or embedded contrast agents which can be interrogated using imaging or spectroscopic means to detect a change in the micro- and nano-topographical complex surface. This is useful for monitoring aspects of the composition when applied or inserted or administered or implanted in a target or patient. The use of embedded contrast agents could provide a means of monitoring the presence, decay, absorption into the target, efficacy of therapeutic effect size.
The following non-limiting examples are illustrative of the present disclosure:
We have developed a cranial window model to study peri-implant healing intravitally over clinically relevant time scales as a function of implant topography. Quantitative intravital confocal imaging reveals that changing the topography (but not chemical composition) of an implant profoundly affects the pattern of peri-implant neovascularization. New vessels develop proximal to the implant and the vascular network matures sooner in the presence of an implant nanosurface. Accelerated angiogenesis can lead to earlier osseointegration through the delivery of osteogenic precursors to, and direct formation of bone on, the implant surface. This study not only highlights an important aspect of peri-implant healing, but also informs the biological rationale for the surface design of putative endosseous implant materials.
We used the model was to determine the outcomes of contact and distance osteogenesis on nanotopographically complex (NT) and machined-surfaced (MA) implants, respectively. Then, we demonstrate that differences in the topography of the surface are reflected in significantly different patterns of peri-implant neovascularization.
All animal procedures conducted in accordance to institutional animal use guidelines approved by University Health Network animal care committee (AUP #4884.0-1). Nine to eleven-week old male C57BL6 mice (Charles River Laboratories, Quebec) were used for the entire study.
The implants were custom-made from grade IV commercially pure titanium, specifically for this study, by ZimmerBiomet Dental, (Palm Beaches Garden, Fla.). The implants were machined from a 4 mm rod stock with a central 2 mm drill hole. Four radially equidistant flutes, with internal radii of 0.5 mm, were machined along the length of the rod. The rod was then machine-sliced, resulting in flat, 4 mm diameter and 500 μm thick, implant forms with the cruciate shape as seen in
Field emission scanning electron microscopy (FE-SEM): Two Ti implants from each surface group were removed from the sterile packs with plastic tweezers and fixed with carbon tape to SEM stubs, taking care to not to damage or contaminate the surfaces. Both the lateral and flat surfaces of the implants were imaged non-coated at an accelerating voltage of 5 keV and increasing magnifications (up to 50,000×) by FE-SEM (Hitachi S-5200, Japan).
X-ray photoelectron spectroscopy (XPS): Implants were analyzed by XPS using a Thermo Fisher Scientific Kα spectrometer (E. Grinstead UK). A monochromatic Al Kα X-ray source was used with a nominal 400 μm spot size. Survey spectra were obtained (200 eV pass energy (PE)) followed by an examination at 150 eV PE of spectral regions of interest from which the relative atomic percentage composition was obtained. High-resolution spectra (25 eV PE) were also obtained for the Ti envelope. Charge compensation was applied for all spectra using a combined e/Ar+ floodgun, and the energy scale was shifted to place the C1s peak at 284.6 eV. All data processing was performed using the Avantage 5.926 software supplied by the manufacturer.
The surgical procedures were performed in a microsurgery room under aseptic conditions on a microsurgical table. Mice were anesthetized with Isoflurane 2.5% vaporized in a 70/30 mixture of O2/N2O. The scalp was shaved and the skin was cleaned with Betadine solution and 70% ethanol. The skin was lifted with tweezers from the midpoint of the ears, cut with fine curved dissecting scissors, and completely removed to expose an 8 mm diameter circular area in the underlying skull. The periosteum was reflected using a periosteal elevator. Once the calvaria was exposed, a midline 4 mm diameter osteotomy was carefully created in the parietal bones using a custom-made trephine (ZimmerBiomet Dental, FL) under continuous irrigation with sterile saline. A guidance stop-line, laser-marked at 200 μm from the tip of the trephine, minimized over-penetration into the craniotomy site during the surgery. The created circular bone piece within the osteotomy was elevated using a periosteal elevator, taking care to leave the dura mater intact. An implant was then placed into the osteotomy. A permanent intracranial imaging window was superimposed over the implantation site to permit imaging through the depth of the healing volumes (and central implant hole), secure the implant, and inhibit the growth of the skin over the defect (
To fix the imaging window, the exposed skull around the osteotomy was first covered with Scotchbond dual cure adhesive resin and then a ring of dental restorative material (3M, Milton, ON) was applied on top of the bonding agent but maintaining a 3 mm distance from the edge of the craniotomy. A round coverslip, 8 mm in diameter, #1 thickness (neuVitro, Germany) was positioned on top of the restorative ring, pressing down gently to secure the Ti implant in the craniotomy. The restorative material was then light-cured to ensure a perfect seal around the defect and to stabilize the coverslip on top of the defect. Physiological body temperature was maintained throughout the surgery and recovery time by a homeothermic pad and healing lamp. Animals were carefully monitored after CIWC placement and they resumed normal activities within 3 days.
Intravital imaging was performed post-operatively to track the microvascular changes during peri-implant healing. Prior to each imaging session, mice were anesthetized by standard intraperitoneal injection of a ketamine/xylazine mixture [80/13 mg/kg]. Each mouse was then administered FITC-DEX (2 MDa; 0.1 mg/mouse; 200 uL injected/mouse) via the tail vein using an ultra-fine 6 mm insulin syringe.
Creation of motion artifacts caused by respiration was controlled and minimized by stabilizing the mouse head on modeling clay and resting the body on a heated stage. In addition, the imaging window was fixed in place by fitting it into a metallic restrainer as demonstrated in
To obtain 3D images of the CIWC, the points above and below the implant in the z-plane were defined by driving the microscope to a point just out of focus on both the top and bottom of the implant surface. Images were recorded as a series of TIF files with dimensions of 1024×1024 pixels. Stacks of images were collected for the FITC channel with Z-stack size≅500 μm. Image acquisition settings were maintained consistent throughout all time points and groups.
Fluorescent images were processed in Zen lite (Zeiss, Jena, Germany) and ImageJ. A MATLAB-based computational code was developed to calculate the functional vessel density. To calculate functional vessel density, maximum intensity projection images of the z-stacks were obtained, binarized and the positive pixel percentage area was calculated for each Region of Interest (ROI).
Quantitative spatial analysis of the vascular network structure in 3-D was performed using the Imaris (ver. 8.3.0, Bitplane AG, Switzerland). The 4 healing volumes represented 4 ROIs that we identified and analyzed at each imaging time point. To measure vessel parameters, each implant healing volume is first oriented in the same manner, as shown in
The animals were euthanized by exposure to CO2 at days 14, 28, or 43 post-surgery. The complete skull was harvested and fixed in 10% formalin for at least 48 hrs. Following fixation, the mandible and the brain were removed, and the dura was kept intact. The samples were further trimmed to remove excess tissue for μCT scanning.
Prepared trimmed samples were scanned using a MicroCT40 (Scanco Medical, Switzerland) at 70 kVp and 114 μA. Images were acquired with a high resolution in three planes, creating slices of 6 μm-thick. A ROI that included the entire defect area was selected, and highlighted in the cross-sectional images from each specimen. ROIs were then reconstructed in 2-D enabling visualization of bone formation in each of the 4 healing volumes in each implant. 2-D images were used as a qualitative demonstration of the mechanism of bone formation (contact vs. distance osteogenesis) at the healing volumes.
Temporal series results (Day 3 to 28) were presented as mean±SEM, and analyzed by two-way repeated measures analysis of variance (ANOVA) in Graphpad. Bonferroni post-tests were performed to test the significance of the means between implant groups at each time point. A confidence level of 95% was considered significant. The in vivo optical imaging procedure was repeated with 6 to 8 animals per time point per implant group. To obtain the statistics of the (vessel) filaments, a D'Agostino-Pearson normality test was performed to assess the normality of all data sets. As the data was not normally distributed, where two implant types were compared at one time-point, Mann-Whitney test was used to assess the statistical significance of the two medians; Interquartile range (IQR) has been shown on the scatter dot Mann-Whitney plots. Where comparing 3 or more groups of data, a Kruskal-Wallis test was performed followed by a Dunn's multiple comparison test. P-values<0.01 were considered significant.
The CIWC was designed to fit precisely into a trephined calvarial defect of 4.0 mm diameter (
The surface topographies of both MA and NT implants were characterized by field emission scanning electron microscopy (FE-SEM). At lower magnifications machining marks were still visible on the MA implants (
To test whether the complex grit blasting, acid etching and nano-tube creation on the NT surfaces induced chemical differences between MA and NT surfaces, we analyzed the elemental composition of the lateral and top surfaces of each type of Ti implant by X-ray photoelectron spectroscopy (XPS). Survey spectra of MA and NT samples are shown in (
Thus, high-resolution Ti 2p spectra were obtained to compare the nature of the TiO2 oxide layer (
Importantly, while we did not detect significant chemical differences between the MA and NT surfaces, they do exhibit differences in their TiO2 surface oxide layer thicknesses and the topographical differences were obvious as observed by FE-SEM.
Samples of the entire skull from both implant groups were scanned at 2, 4 and 6 weeks after implantation using microcomputed tomography (μCT). No bone was detected in the healing volumes at week 2 in the NT group (Supplementary
The spatio-temporal dynamics of peri-implant wound healing were examined in vivo in C57BL6 mice using our experimental CIWC model. The CIWC remained durable, and infection-free, for up to at least 6 weeks. The CIWC permitted intravital longitudinal tracking of neovascularization at the peri-implant wound site by confocal fluorescence microscopy. Vessels were visualized by tail vein injection of a high molecular weight (2 MDa) fluorescein isothiocyanate-dextran (FITC-DEX) that had a low extravasation rate in intact vessels. Development of the vasculature in the peri-implant healing site was tracked from day 3 to 42 post-implantation. Neovascularization occurred earlier around the NT surface than the MA surface, and extravasated FITC-DEX was mostly visible from the vessel tips at earlier time points (
Comparative analysis of the functional vessel density28,29, from weeks 1 to 6, was quantified (as % fluorescent area of each defect) by keeping the concentration and administration dose of FITC-DEX the same in both implant groups, and across all imaging time points (
To characterize the morphology of the vasculature developed proximal to the implants at early time points after implantation, morphometric analysis was performed on the confocal intravital images of the FITC-DEX taken at days 7 and 15 post implant surgery. An example of an healing volume from each of the MA and NT groups, respectively, is shown in
At day 7, the NT group exhibited a hierarchically branched network of the vessels with small branches that grew over the surface of the implant and distributed along the lateral surface (
While neovascularization is an essential prerequisite to osteogenesis, no previous published reports have examined the effect of implant surface topography on the spatiotemporal pattern of neovascularization during endosseous peri-implant healing in vivo. Our results clearly show that the surface design of the implant has a profound effect on the pattern of neovascularization with new vessels being developed at, or near, the implant surface and the vascular network maturing through remodeling sooner in the presence of a topographically complex surface. The rapid development of a functional vascular supply is of key importance to pen-implant wound healing, both as a source of scavenging and immune-modulating leukocytes, and a nutrient supply to support tissue regeneration. Indeed, the rate of osseointegration is critically dependent upon osteoconduction—the key determinant of contact osteogenesis30—and we have shown, quantitatively, that this can be accelerated by increasing the topographic complexity of the implant surface31. Thus, our findings provide a new perspective on the importance of implant surface design that is relevant to many therapeutic areas including orthopedics, dentistry, otorhinolaryngology and plastic surgery. Previous studies have established the window chamber model as a tool to longitudinally image the spatia-temporal development of both neovascularization and osteogenesis in craniotomies32,33. An observation common to these, and microCT, calvarial studies is that new bone grows centripetally within the bony defect both in the un-modified state34,35 or when the defect is modified by the addition of growth factors34,35, cells36 or cells and scaffolds36-38. This is important because we show, on the contrary, that when a metallic implant is introduced into such a model, the pattern of bone growth is modulated as a function of implant surface topography: the MA (smoother) and NT (rougher) surfaces exhibited distance and contact osteogenesis respectively1. This observation provides an essential validation of our CIWC model as it has been generally accepted, since the work of Buser et al (1991)12, that implant surface topography has a profound effect on contact osteogenesis. Indeed, we have established the functional significance of three distinct scale ranges of implant topography on both bone bonding and bone anchorage, two distinct mechanisms within the phenomenon of osseointegration39. The current study investigated the effect of implant surface topography on peri-implant neovascularization using two surfaces, a relatively smooth machined (MA) surface and a complex microtopographic surface with superimposed nanotubes (NT). However, our platform would be suitable for studying spatia-temporal vascular morphogenesis around other surfaces beyond those discussed in the present paper.
Our model has enabled direct visualization of three distinct phases of vascularization during the first 42 days of healing: capillaries sprouted and grew longer, anastomosed to form loops and, finally, remodeled into a more functional vasculature that facilitated blood flow throughout the peri-implant site. High-resolution images showed that the vasculature grew predominantly from the periphery of the bony defect towards the lateral surface of the implant, but vessels also grew from the dural surface into the central implant hole. With time this peripheral and central vasculature anastomosed on the top flat surface of the implant, with blood flow in each direction (Supplementary Movie 1). Although such anastomoses occurred on both the machined and topographically complex surfaced implants, only the latter displayed an ordered, radial, arrangement of vessels, a pattern completely absent on the machined surface, during the time course of our experiments. Indeed, we demonstrated that the NT surface not only increased the rate of neovascularization following endosseous implantation, but also changed the morphological characteristics, spatial pattern, and functionality of the re-established microvasculature. Interestingly, while the machining marks were obvious on the machined implant, they were less evident on the complex surface. There have been numerous reports of cell migration along the long axes of surfaces with linear features40,41 but we saw no evidence that these topographic features influenced the directional growth of vessels.
At the earliest days of healing, in both implant groups, the neovessels were highly permeable as FITC-DEX extravasated from both the lumen and ends of the nascent vessels, appearing as a bloom of fluorescence. With time, and increasing function, extravasation of the FITC-DEX through the vessel walls was reduced and only leaked out from the vessel tips. We believe that such extravasation is due to the immaturity of the distal blood vessels, since it was absent at later time points.
Morphological properties of the microvascular system affect the blood flow and its distribution within the wound area42. The morphometric parameters used in this study which were measures of vascular density, volume, length and branching number are indicators of the ability of the vasculature to distribute flow throughout the tissue. These are standard parameters used by several studies assessing physiological32,43 or pathological angiogenesis44,45, although representation of the data on combined box/whisker and scatter plots provides additional graphic information concerning the frequency distribution of the individual vessels in the complex 3D network.
From the physiological standpoint, distribution and collection of blood-borne substances within tissues and organs requires a branching system. Hierarchical branching of a vascular network—starting from a relatively large stem vessel to smaller and smaller branches—is essential for conducting flow further into the wounded area. However, a non-optimal vascular density reduces vascular functionality46. Therefore, the pruning of excessive vessels is essential for maturation of a vascular network. The branching number shows increased branching around NT implants compared to MA implants. The early dense network of small vessels matures, through remodeling, to larger functional vessels that conduct a higher volume of the blood. During the maturation of the vascular network some of the morphological features such as vessel length, volume and branching change concomitantly as there are scaling relations between these parameters47. The choice of one vessel over another in the pruning process, is known to be based on blood flow48. Vessels with higher blood flow increase in girth while those with lesser blood flow regress. Our results show a higher mean vessel volume in the NT group both at week 1 and 2 compared to the MA group. However, the number of vessels is higher in the MA group. This indicates that large vessels have an essential role in increasing the bulk flow compared to numerous small vessels. By week 4, the vascular network was remodeled to form larger vessels that improved functional blood flow for both implant types. This measure of blood flow is important since it has been reported that the progenitor cells position themselves relative to the volume of the blood49, and vessels were consistently larger around the NT implants.
Since we would not expect to image vessels that may have formed independent of the pre-existing vascular network, as they would not be labeled with FITC-DEX unless they had anastomosed with those that had developed from the functional vasculature of the circulation, we cannot exclude the possibility of vasculogenesis as distinct from angiogenesis50. However, our results do show that the changing characteristics, structural organization, and spatial location of the re-established vascular network around the two implant surfaces was reflected in a corresponding change in the spatial pattern of bone healing. Previous cranial defect healing models have suggested that the osteogenic precursor cells can originate from the periosteum, bone marrow (BM)51,52 and dura matter53, and we would expect these tissue-resident mesenchymal cells, to be of perivascular origin54 although not pericytes55. In fact, Hung et al. showed that there is a correlation between the morphometric characteristics of the vascular network, particularly the diameter and the length of the blood vessels and the volume of the differentiated osteoblasts in their vicinity56. Thus, by altering the surface characteristics of the implant, which we have shown to have profound effects on neo-vascularization, the ingress of osteogenic precursors and their location with respect to the implant surface is also being affected, resulting in either contact or distance osteogenesis.
In contradistinction to previous reports, our model provides a unique and reproducible preclinical platform to study implant healing biology over clinically relevant time scales. The window is durable for more than 6 weeks, sufficient to monitor early critical stages of both peri-implant neovascularization and osteogenesis. Using intravital imaging, we obtained both qualitative and quantitative information on the complex 3D structure of the neovascularization with respect to the two different implant surfaces over a large region of interest (4 mm). Tracking active vascularization from initiation to remodeling in a single animal, over multiple time points, reduces animal-to-animal variation and increases the reliability of the quantification. Interestingly, the presence of the implant blocked much tissue auto-fluorescence and resulted in an increased signal-to-noise ratio. Together with longer pixel dwell, these details may account for the higher resolution images we obtained compared to previous intravital studies32,33. Titanium-based implant materials are commonly employed in orthopedic, craniofacial and dental surgery due to their combination of mechanical properties, corrosion resistance and biocompatibility57-59. Our results show that a topographically complex surface contributes to the development of a radially arranged vascular structure with hierarchical branches spatially closer to the surface of the Ti-implant. These findings emphasize the translational importance of a rationale for implant surface design, which could help improve the clinical effectiveness of endosseous implants compared to traditional implant surfaces. As neovascularization is the route for the ingress of both immune and progenitor cells, alterations in the surface topography would enable healing through regulation of neovascularization. A comprehensive understanding of the healing and regeneration mechanisms of endosseous integration in the pen-implant niche has a considerable impact in implant medicine. The knowledge transferred from the current study provides one step forward towards designing endosseous implants capable of controlling endogenous peri-implant vascularization.
We have recently developed a cranial implant window model4 with which we have longitudinally tracked the spatia-temporal development of peri-implant neo-vasculature using intra-vital microscopy5. Using this model, we have shown that the pattern of angiogenesis in the wound site can be profoundly, and reproducibly, influenced by the surface topography of a metallic implant. Since angiogenesis precedes osteogenesis in bone wound healing, the model enabled us to demonstrate that the pattern of peri-implant angiogenesis determined that bone formed in contact with a topographically complex (TiNT), but distant from a smoother machined (TiMA), titanium implant surface. However, the means by which the mesenchymal osteoprogenitors populated the wound site were not examined.
Now, using the same implant surfaces, applying our imaging model to a Hic1 (Hypermethylated in Cancer-1) mouse model, in which perivascular mesenchymal progenitor (MPs) cells are labeled with a fluorescent protein (tdtomato), has allowed us to longitudinally track MP migration, the differentiation of their progeny, and their spatiotemporal relationships to neo-vascularization of the wound site.
Hic1 is a gene involved in craniofacial development6,7 in both human and mouse and marks a broad population of perivascular mesenchymal progenitor cells8. The Hic1 marker extensively overlaps with PDGFRα and Sca18, which are common markers of mesenchymal precursors in various tissues9-11. PDGFRα is a mesenchymal marker in both human and mouse; and it was recently found that PDGFRα+ cells that reside in injured peripheral nerves are mesenchymal precursors that can directly contribute to digit tip regeneration and skin repair in mouse12. On the contrary, Sca1 is a marker of hematopoietic and mesenchymal cells unique to mouse. Thus, we assessed the impact of implant surface topography on MP ingress into the pen-implant healing compartment.
Increasing implant surface topographic complexity results in enhanced platelet activation13 and consequent signaling. We hypothesized that the migration of both perivascular and endothelial cells could be driven by the differential activation of platelets on the implant surfaces employed. To test this hypothesis, we undertook in vitro modeling to interrogate the migratory behavior of both Hic1+ and endothelial cell populations in the presence of a linear density gradients of human platelet lysate (PL).
Intravital Imaging Reveals that Complex Implant Topography Enhances Recruitment of Mesenchymal Progenitor Cells to the Implant Surface. (
We assessed the dynamics of peri-implant MP ingress by repeated intravital imaging. We studied the behavior of MPs intravitally in mice containing tdTomato reporter knocked into the Hypermethylated in cancer (Hic1) gene. We crossed a (Hic1-CreERT2) knock-in line with RosaLSL-tdTomato mice. Tamoxifen injection for five consecutive days gave rise to strong Hic1-specific expression of tdTomato. After a 10-day wash-out period, live imaging was performed according to the timeline illustrated in
d and e are representative images taken through the window chamber implanted within the calvaria of Hic1/tdTomato mouse. Three fluorophore channels were imaged by confocal microcopy; endogenously fluorescent MPs (tdTomato) in red, new functional blood vessels visualized by tail vein injection of high molecular weight (2 MDa) fluorescein isothiocyanate-dextran (FITC-Dex) in green, and the titanium implant (silver gray) within the bone. Images were collected in tiled z-stacks of the entire implant wound area, which contains a titanium implant placed in a 4 mm in diameter craniotomy.
Mesenchymal Progenitor Cells are Perivascular, but not Pericytes, and Their Entry in the Wound Site is Correlated with Angiogenesis (
To elucidate the events occurring within the bone-implant wound site, we closely looked at one of the healing volumes in the cruciate shaped implant. We performed intravital longitudinal imaging on Hic1/tdTomato mice from day 3 to 42 post-implantation and tracked the healing events in the proximity of the implants with nano (TiNT) and smooth (TiMA) surfaces (
Longitudinal observation of tdTomato expression demonstrates dynamic changes in the population of MPs over time. What was remarkable here was the emergence of a proliferative bloom of tdTomato+ cells in the TiNT group at day 7 post-implantation which peaked at day 11, and diminished by day 28. Simultaneous imaging of the MP cells and the blood vessels suggests an association between the population of the MPs in the wound site and the growth of the new vessels. Interestingly, the population of tdTomato progenitor cells and blood vessels was remarkably lower at all early timepoints in the TiMA group compared to the TiNT group. The appearance of leaky vessels indicates slower development and maturity of blood vessels at day 7 post-implantation in the TiMA group. The absence of the bloom of tdTomato cells in the periphery of the TiMA implant is an indication of lower regenerative activity. The early progression of neo-vascularization clearly visualized at Day 7 around the TiNT implant was only seen at Day 28 in the TiMA samples, at which time vascular remodeling around the TiNT samples was evident.
The quantification of functional vessel density and the number of the MPs present in a healing volume at early timepoints (day 3 to day 11) for both implant groups (
Despite extensive evidence of an association of mesenchymal progenitor cell recruitment with wound site vascularization, it appears that these perivascular cells are not bound to the newly growing vessels in the first few days post-implantation. In
3D spatial analysis of red (Hic1+ MPs) and green (blood vessels) fluorescence channels was performed to identify the microanatomical location of activated MPs in the peri-implant wound site. 3D reconstructions of each implant healing volume were created as demonstrated in
The above discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.
We identified the original location of the tdTomato/Hic1 positive cells within the cranium and compared the quantity of these cells and the vasculature in injured versus intact cranium. For both conditions, histology confirmed the presence of the tdTomato cells in the periosteum, diplöe, and dura mater, but the majority of MPs were visible in the inner layer of periosteum (
Intravital Microscopy (lVM) on single cells in the cranial defect at various timepoints during healing identified phenotypically distinct cells labeled with tdTomato (
Human umbilical cord perivascular cells (HUCPVCs) were used for all in vitro experiments as a characterized population of perivascular cells. These cells are a non-hematopoietic population of cells isolated from the perivascular tissue of the cord that are capable of differentiating into myogenic, adipogenic, chondrogenic, and osteogenic lineages in vitro16. These cells have a fibroblast-like morphology with a stellate shape and long cytoplasmic processes. Flow cytometry showed the expression of MSC surface markers CD105, CD90, CD73 CD166, CD146, CD 140b/PDGFRb, CD10 and MHC1. Also, these cells lacked the expression of hematopoietic lineage markers CD34, CD45 and HLA-DR and endothelial cell markers CD31 (
Our previous in vivo assays showed that the nanosurface changes the pattern of neovascularization in, and the current findings the recruitment of MPs to, the peri-implant compartment. The cause of this differential pattern is currently unknown. However, platelets activated on the implant surface release multiple growth factors and cytokines (i.e. PDGFα, PDGFβ, VEGF-A and TGFβ) in high concentrations and provide ligands for the cells that reside in the wound niche. Therefore, we conducted in vitro tests for the real-time chemotaxis and migration of endothelial and Hic1+ MPs in response to a linear gradient, at various concentrations, of human platelet lysate (PL). Both cell types adhered to the bottom of the slide (
In this study we showed that the microvascular bed contains resident mesenchymal progenitor cells that directly contribute to post-surgical tissue repair and regeneration. We used IVM to anatomically and functionally map blood vessels and associated mesenchymal cells. We first showed that the Hic1+ MP cells populate the wound site rapidly to form a dense “bloom” of proliferating cells which appear in areas demarcated by the neo-vasculature but show no preferential juxta-positioning to the vessels themselves. Interestingly within the time frame of 11 to 28 days, the number of the tdTomato labeled Hic1+ cells diminishes but an increasing number of these cells are found in intimate contact with the vasculature displaying a pericytic morphology with cell processes wrapping around individual vessels or vessel junctions. Similarly, other Hic1+ cells exhibit either a fibroblastic migratory morphology or become incorporated in the forming bone tissue as osteocytes.
Using the same mouse model of Hic1CreERT2/tdTomato, Soliman et al. have shown that Hic1+ cells are a heterogenous population of progenitor cells having two main subclusters of PDGFRa+/Sca1+ and PDGFRa+/Sca1−. The PDGFRa+/Sca-1+ subtype is a multipotent population of mesenchymal progenitors and upon injury some of the Sca1+ differentiate into Sca1− subtype17. The anatomical locations of PDGFRa+/Sca-1+ and PDGFRa+/Sca-1− cells were not clearly addressed in the myocardium by Soliman et al. However, in the cranium, there are three obvious sources of cells: the periosteum, the diplöe, and the dura mater also known as the endocranial periosteum. In our study, labeling was induced at post-natal week 8 following tamoxifen treatment, immunolabeling of tdtomato+ cells at week 14 post-labeling showed the presence of these cells in the periosteum, the outer layer of dura mater, and a few labeled cells in the diplöe. It should be noted that by 14 weeks the diplöe is a protected environment since the inner and outer tables of the cranium have closed at the osteotomy site (
Together, these observations suggest that tissue-resident mesenchymal progenitor cells are localized between capillaries and enter the wound site where regeneration is needed along with capillary growth. However, this proliferating population of tissue resident progenitor cells are not pericytes, as initially thought to be in multiple organs including skeletal muscle. In fact, several studies have identified pericytes as tissue-resident progenitor cells in multiple human organs18-20 by their expression of CD146, NG2, and PDGFRβ, and absence of hematopoietic, endothelial, and myogenic cell markers. Recently Guimarães-Camboa et al. (2017) challenged this premise by discovery of a novel gene, Tbx18, exclusively expressed by mural cells of adult organs. Their study indicates that PDGFRβ is not a reliable marker for pericyte lineage tracing21. A lineage tracing study using a Tbx18-CreERT2 mouse line revealed that despite obtaining promising in vitro data, pericytes and vascular smooth muscle cells (VSMCs) did not display endogenous multi-lineage potential during aging and injury. However, Guimarães-Camboa's findings are exclusive to mural cells and do not exclude the possibility of other cells existing in the perivascular niche that might have progenitor properties21. The findings of their study are aligned with our in vivo microscopical observations. However, they are in contradiction to what has been described by Diaz-Flores22, who contends that pericytes, specialized cells sharing the basement membrane with endothelial cells, are activated and separated from walls of the blood vessels to become transitional cells and ultimately differentiate into osteoblasts.
The divergence of these results helps distinguish between pericytes—cells sharing a basement membrane with endothelial cells—and perivascular cells, the cells that reside in the vicinity of the blood vessels. Specifically, the appearance of morphological distinct pericytes at later time points, and their absence in the blooming tDTomato population at earlier tiome points in our own work, would indicate that the progenitor population is not pericytic. However, it is clear that mural and perivascular cells of different organs are heterogenous populations, and thus the multipotency of these cells23 at various developmental stages, or adult healing conditions, should be explored in their native environment.
Interestingly, our model shows that a massive proliferating bloom of MPs was observed in the recipients of the topographically complex implant, and the total number of tdTomato MPs was significantly higher compared to the smoother machined implant. This observation strengthens the notion that implant surface is the regulator of the extent of regeneration. The driving mechanism may be explained, in part, by our trajectory plots of individual cells in vitro, which showed that exposure to a local PL gradient not only provided stimulus for migration and recruitment of both endothelial and perivascular cells, but also controlled the directionality of migration for both cell types. Thus, during peri-implant healing the formation, and direction, of blood vessels5, is spatiotemporally associated with the ingress of mesenchymal progenitors to the wound site.
All animal procedures conducted in accordance with institutional animal use guidelines approved by University Health Network animal care committee (AUP #4884.1-2), Toronto, Ontario Canada.
Hic1CreERT2 mice were sourced from the laboratory of one of us (TMU) at the University of British Columbia, Canada. For lineage tracing purpose, Hic1CreERT2 mice were interbred in-house with RosaLSL-tdTomato mice (The Jackson Laboratories stock #007914) to generate a mouse colony expressing tdTomato HiC1+ mesenchymal progenitor cells (MPs). To induce CRE-ERT2 nuclear translocation, 8 weeks-old mice were administered 100 μL per day intraperitoneally with 30 mg/mL of Tamoxifen in sunflower oil for 5 consecutive days. A 10-days washout period was considered before the mice were ready for experiments.
Cranial implant window chamber placement surgeries were performed on mice and Intravital images were acquired using an LSM 710 (Zeiss, Germany) according to a previously described protocol14. Prior to each imaging session, mice were anesthetized by intraperitoneal injection of a mixture of Ketamine and Xylazine. Each mouse was administered 200 μm FITC-DEX (2 MDa; 0.1 mg/mouse) via the tail vein to visualize microvasculature (488 nm excitation, 500-550 nm emission). The imaging procedure was followed according to the experimental timeline shown in
Image processing and analysis of the intravital confocal images was performed in Zen lite (Zeiss, Jena, Germany) and Imaris (ver. 8.3.0, Bitplane AG, Switzerland). Functional vessel density was obtained by calculating the positive pixel percentage of a z-stacks. Quantitative spatial analysis of cells in the in the peri-implant wound site has been performed using Imaris spots. The 4 healing volumes represented 4 ROIs that we identified and analyzed at each imaging time point.
A 4 mm osteotomy was performed in the cranium of 10-week old Hic1/tdTomato mice. Implant-free window chambers were placed following the same protocol for CIWC placement. The mice were euthanized at day 42 post-implantation. The cranium was collected and assessed by ex-vivo histology for RFP (tdTomato), CD31 (blood vessels), as well as hematoxylin and eosin (H&E). Intact cranial bone collected from same age mice served as controls.
The animals were euthanized by exposure to CO2 at days 43 post-surgery. After dissecting the skull, the mandible and the brain were removed. The samples were further trimmed to remove excess tissue and fixed in 4% Paraformaldehyde (PFA) for at 24 hrs. For histology, samples were decalcified using 14% EDTA solution in distilled water and embedded in paraffin. Coronal sections (6 μm) were obtained from the middle of the defect, consecutive slides were then stained for hematoxylin & eosin, Red fluorescent protein (RFP) (Abcam Cat. No. ab34771) for tdTomato cell, and CD31 (Abcam Cat. No. 28364) at 1:400 and 1:50 diluted in dako diluent (Dako Cat. No. S0809) for endothelial cells. Images were acquired using Aperio AT2 whole slide scanner (Leica, Canada) at 20× magnification.
Perivascular cells: human umbilical cord perivascular cells (HUCPVCs) isolated by physical extraction from umbilical cord vessels under a procedure performed by Tissue Regeneration Therapeutics Inc (Toronto, CA), followed by explant culture of perivascular tissue in serum-free conditions, passaged (at seeding density of 1,333.33 cells/cm2) and harvested at day 5 and 80% confluency at passage #3. TheraPEAK™ MSCGM-CD serum-free Mesenchymal Stem cell Growth Medium (Lonza; Cat. No. 00190632) medium was used for culture, which was changed every 3 days. TrypLE Select CTS (lnvitrogen; Cat. No. A1285901) was used for enzymatic dissociation at 80% confluency. HUCPVCs used for all in vitro migration and chemotaxis assays were pooled from 5 different donors.
Endothelial cells: human umbilical vein endothelial cells (HUVECs) were obtained from Tissue Regenerative Therapeutics Inc (Toronto, CA). Cells were cultured in Endothelial growth medium-2 (Lonza; CC-3162) supplemented with 2% serum and 1% Antibiotics at a seeding density of 0.1×105 cells/cm2. The medium was changed the day after seeding and every other day thereafter. Cells were pooled from 3 different donors and were harvested at passage 3 at 70-80% confluency for migration and chemotaxis experiments.
BM-MSCs: human BM-MSCs were provided by Tissue Regeneration Therapeutics Inc. Culture conditions and techniques were the same as described for HUCPVCs.
Flow cytometry: Briefly, 1×105 frozen-thawed HUCPVCs were washed in PBS containing 1% BSA and 2 mM EDTA (flow buffer) and incubated for 30 minutes at 4° C. in the same buffer containing the following conjugated anti-human antibodies (at 1:5-1:20 dilutions): HLA-DR-FITC, CD31-FITC, CD45-FITC, CD10-FITC, CD142-APC and CD34-APC (eBioscience); CD90-FITC, CD73-PE, CD105-PE, CD166-PE, CD146-PE, CD140b-PE and MHC I-APC (BD Biosciences). The cell suspensions were then washed with flow buffer and resuspended in flow buffer. Immediately before analysis on the Cytomix FC 500 flow cytometer (Beckman Coulter), cells were stained with Propidium Iodide (PI) to exclude dead cells and 5000 live or PI-negative events were collected. Surface marker detection via antibodies was measured in FL1 for FITC, FL2 for PE and FL4 for APC. Flow cytometry data were analyzed using Kaluza Software (Beckman Coulter) and presented as a positive % expression or mean fluorescence intensity (MFI) which is a measure of the intensity of the signal.
Microarray: HUCPVCs and BM-MSCs were cultured on 6 well plates using the conditions previously described. The RNA was isolated when reached 80% confluency using Tri Reagent (Sigma) and later purified using RNeasy MinElute cleanup kit (Qiagen, Canada) as per manufacturer's instructions. RNA purity and yield were determined using the NanoDrop 1000 (Thermo Fisher Scientific, Wilmington, Del.), and quality with Agilent 2100 bioanalyzer (Agilent Technologies, Canada). 8 HUCPVC biological replicates and 7 BM-MSC biological replicates were used for microarray analysis using the GeneChip Human Gene 1.0 ST array (Affymetrix, Santa Clara, Calif.) as per manufacturer's instructions.
Real-time chemotaxis assay: chemotaxis of HUVECs and HUCPVCs towards various concentrations of the platelet lysate was analyzed in real-time using 2D chemotaxis u-slides (ibidi, Germany). Cells suspension in complete medium harvested at passage 3, were seeded in the observation area of the μ-slide according to the protocol provided by the supplier at a concentration of 1.5×106 cells/ml. Chemotaxis μ-slide (ibidi GmbH) forms a diffusion-based gradient of soluble growth factors. Each setup is 10 mm wide and 200 um high. There are 3 setups on each slide with the size of a microscope slide. There are 3 channels in each setup. A narrow observation area connects two larger reservoirs. The cells are initially seeded in the observation area, the left reservoir is filled with chemoattractant and the right reservoir is filled with a culture medium. By diffusion, cells will be exposed by a linear gradient that will stay stable for 48 hours24. The μ-slides were incubated at 37 C.° for 2 hours to allow enough time to the cells to adhere to the bottom of the device. The μ-slides has two reservoirs to either side of the observation area. The left reservoir was filled with 25, 50, 70% Platelet Lysate (PL) (Cook Medical, Bloomington, Ind.) in complete medium and the right reservoir with the complete medium. By diffusion, cells will be exposed to a linear gradient of a PL. Time-lapse video microscopy was conducted using inverted live cell microscope (Zeiss, Germany) with 4× objective for 24 hours to observe the chemotaxis of the cells in response to a linear gradient of platelet lysate. Cell tracking was performed with the manual tracking plugin in ImageJ. Chemotaxis and motility parameters, including forward migration index (FMIx, FMIy), mean velocity, and P-value of the Rayleigh test, were calculated and plotted using ibidi chemotaxis tools.
The Boyden chamber migration assay was performed using Transwell inserts with 8 μm pores (Corning, N.Y.). A total of 50,000 in 200 μL cells were added to the top compartment of the inserts, which were placed into 12-well plates. The filters were transferred into wells containing 1000 μL 0, 10, 25, 50, 100% Platelet lysate (PL) in serum-free Lonza medium (SFM). The filters were collected from the well-plates after 12 and 6 hours of incubating at 37° C. for HUCPCs, and HUVECs respectively. Filters were fixed with 4% paraformaldehyde (PFA) and stained with Hoechst 33342 for nuclei. The number of cells that transmigrated to the underside of the filters were counted in each well using an inverted fluorescence microscope.
Temporal series results (Day 3 to 28) were presented as mean±SEM and analyzed by two-way repeated measures analysis of variance (ANOVA) in GraphPad. Bonferroni post-tests were performed to test the significance of the means between implant groups at each time point. A confidence level of 95% was considered significant. The intravital procedure was repeated with 6 to 8 animals per time point per implant group. P-values<0.01 were considered significant.
While the present application has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the application is not limited to the disclosed examples. To the contrary, the application is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2019/050853 | 6/17/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62686067 | Jun 2018 | US |
