RS20190610A1 - Osteoinductive bone graft based on nanohydroxyapatite and copolymer of lactic and glycolic acid (polylactide co glycolide)-plga for repairing of bone tissues of long bones - Google Patents

Abstract

The present invention relates to a process for obtaining osteoinductive resorbable 3D grafts based on NanoHA and PLGA / PLA as bone substitutes, by the method of extrusion printing. Such grafts have potential application in the reparation of super-critical defects and bone defects of large dimensions with interruptions of tubular bones. Graft design ensures a remarkably high porosity and excellent mechanical characteristics, which makes it applicable also for large bone defects of the bones which are under load, which is a unique success. In addition, it possess extraordinary good osteoinductivity, allowing a rapid transformation of the graft / implant in a newly formed bone tissue, which was confirmed by numerous histological analyzes. Additionally, the non-toxicity of these grafts was approved by using a wide scale of various in vitro (assays on the L929 fibroblasts and stem cells derived from dental pulp, genotoxicity assay on human lymphocytes) and in vivo assays, (cutanous and subcutanous irritation, acute toxicity) including assay of long-term toxicity. This opens the way for its potential clinical application in various branches of implantology, from maxillofacial surgery to orthopedic and reconstructive surgery.

Description

Osteoinductive graft based on nano-hydroxyapatite and copolymer of lactic and glycolic acid (poly(lactide-co-glycolide) - PLGA) for the repair of bone tissues of long bones

Description of the invention

Osteoinductive bone substitutes in bone tissue engineering, which enable the repair of critical bone defects, especially defects related to the replacement of parts of damaged long bones, are described in this patent. Bone substitutes are based on a composite system consisting of nanohydroxyapatite (nano-HA) and PLGA, where the supporting part of the structure is made of HA, while the surface layer of the granules is covered with a thin film of PLGA. As a temporary polymer support (template), polymer fibers in the form of a sieve of appropriate diameter and polymer fibers as a dispersing element were used, which, with their dimensions, enable the continuity of the pore network within the HA support. In addition, dispersing elements and polystyrene polymers with a precisely defined particle size distribution were used, in order to ensure the multimodality of the dispersion phase, which was then removed in different ways: by dissolution or burning, with subsequent sintering, whereby a granulate was provided that, after being covered with a thin film of PLGA, was suitable for 3D printing. 3D printing was performed by using an X-ray or micro CT image of the part of the bone that needed to be replaced as a model, in order to use the appropriate software through the gradient layering of nano-HA and PLGA composites, using the extrusion printing method, obtained a completely adequate part with the part of the bone that needs to be replaced. The nanostructural design of HA with a high degree of activity and well-adjusted dissolution rates of the crystal lattice, which were optimized to the value of biological apatite, gave such constructs not only adequate mechanical characteristics, but also exceptional osteoconductivity, which enabled the newly created bone to be created at the rate at which the implant itself is dissolved, which was of key importance for achieving efficient and rapid osseointegration, through the process of complete replacement of the implant with the newly formed bone.

The technical field to which the invention belongs

This invention relates to the technological process of designing complex bone grafts/implants that serve for the regeneration of bone supercritical defects and bone defects caused by interruption of the continuity of long bones. More precisely, the invention relates to the design of bioinductive bone implants that can withstand natural loads, while being transformed into natural bone, which in its characteristics is close to the characteristics of natural bone.

The technical problem that the invention solves

Bone repair and reconstruction is an important clinical problem. Depending on the type of clinical application, it is estimated that some of them are extremely common and that, as in periodontology, they include very broad layers of the population, even up to several percent, while more complex reconstructions of bone tissues include several per thousand. Of course, bearing in mind that with osteoinductive materials, such as the material that is the subject of this patent, it is possible to repair a significantly larger volume of bone defects, which include the replacement of cancerous bone tissues, as well as reconstructions aimed at reconstructive and plastic surgery; then the volume of new applications increases significantly, significantly multiplying the possibilities of applying this type of implant.

State of the art

Traditional biological procedures, which include cancellous bone autografts and allografts, are mostly based on the application of vascularized grafts from the fibula and iliac crest. In today's time of increased use of bone grafts, the failure rate is very high and ranges from 16% to 50%. The rate of failed autografts corresponds to the lower limit of the specified interval. Nevertheless, due to the limitations in the possibility of applying autografts, there is a very high demand for donor grafts, but various issues arise such as: the limitation of the amount of such grafts, their adequacy of shape and size, the unpredictability of the speed of bone repair, the problematic speed of resorption. In the case of larger defects, complete resorption of the bone graft occurs before the osteogenesis process is completed. The time it takes to extract an autograft is very expensive, and tissue donors are often rare. Many donated tissues taken from deceased individuals are associated with infection and hematoma. Allografts carry the risk of disease and/or infection, which can cause a weakening or complete loss of bone inductive factors. Vascularized grafts require a serious microsurgical operative procedure, which requires a sophisticated infrastructure. Techniques of interfering with osteogenesis are often difficult and long-term processes, which require high motivation of the patients.

Bone tissue engineering using cells in combination with synthetic extracellular matrix is a new approach in relation to tissue transplantation. Numerous tissue engineering concepts related to the design of new bone grafts have been proposed. Such solutions attempt to mimic the success of autografts, by removing cells from the patient via biopsy, followed by the growth of a sufficient amount of mineralized tissue in vitro to then perform the implantation procedure, in the form of a 3D scaffold for use as functionally equivalent autogenous bone tissue. In this way, the essential properties of autogenous bone material are reproduced, creating an ideal environment for bone tissue regeneration, which includes the following characteristics: highly porous material with 3D architecture that allows osteoblastic and osteoprogenitor cell migration and graft revascularization; the ability to bond with the surrounding host bone and resume the normal process of bone remodeling; normal proliferation of bone cells, and osteogenic growth factors for accelerated healing and differentiation of local osteoprogenitor cells.

Grafts based on natural or synthetic materials inside the body create temporary structures, which allow the body to use its own cells to form new bone tissue while the scaffold is gradually absorbed. Conventional two-dimensional scaffolds are satisfactory for cell proliferation, but are much less satisfactory when it comes to generating functional tissues. For this reason, three-dimensional (3D) bioresorbable and especially osteoinductive scaffolds are desirable for the generation and maintenance of highly differentiated tissues. An ideal scaffold should have the following characteristics: highly porous structure, with interconnected pores for cell growth and transport of nutrients and metabolic waste; to be biocompatible and bioresorbable, with a controlled rate of degradation and resorption and that this rate essentially coincides with the rate of new tissue formation; to possess appropriate surface chemistry suitable for cell attachment, proliferation and differentiation; to possess sufficient channels to promote vascular integration and to possess mechanical properties similar to those of the tissue being replaced. In vivo, the interior of the pore network scaffold should be protected from proliferative cells and their extracellular matrix for a sufficiently long time. This is especially important for load-bearing tissues such as bone and cartilage.

A biomechanically stable scaffold with extremely high open porosity and adequate multimodal pore size distribution is crucial for new bone formation in vivo. Repair and reconstruction of large bone defects, as well as lengthening of the lower extremities, is a clinical challenge of a special kind. This problem has not been solved yet. Currently, none of the proposed approaches have demonstrated long-term efficacy that resembles natural bone. A suitable scaffold has yet to be designed. Ceramic materials are brittle and scaffolds are prone to premature breakage. In many cases, HA ceramic is very poorly resorbable, because the resorption period lasts more than 6 years. Bioresorbable polymer scaffolds have been used but none have been designed for long bone tissue applications. These high glass transition temperature polymers such as polyglycolic acid (PGA) and polylactic acid (PLA) are brittle and some degrade too quickly, so they are unable to induce the long-term mechanotherapeutic induction necessary for a proper bone remodeling process. The formation of acid in high concentration during their degradation, in the case of polymers with too short a degradation time, also prevents the proper formation of cells and bone growth. Another problem is that large-sized scaffolds do not have enough channels to provide a rich vasculature of blood vessels for the removal of waste products, as well as the delivery of nutrients throughout the circumference. Scaffolds with high porosity also lack sufficient mechanical stability to be used for structured bone tissue in long bone engineering.

Exposing the essence of innovation

The subject invention describes a process for the fabrication and design of a tubular scaffold pore, which has a central channel and multiple side channels that are resistant to breakage and possess sufficient flexibility under bending, compression and torsion forces. The design that has a central channel allows easy proliferation of various constituents such as cells, tissues and their products, growth factors, drugs and their combinations, while side channels along the entire circumference of the scaffold allow the communication of blood vessels. The construction consists of two layers: an internal porous part that has a central channel with micro-channels along the plane of the surface, which provide the primary site for osteogenesis and provide adequate compressive strength; and an outer porous part that has microchannels along the central axis, which enable cell proliferation, vascular integration as well as torsional and bending stability. During the design of such a graft, the proportion of porous bioresorbable polymer in the nano-HA composite with adequate biosolubility was increased, so that in the inner part there was mostly a nanocomposite with a significantly smaller proportion of polymer. Scaffold is a network of nano-HA granules with extremely high porosity and resorbability that is adjusted to the resorption of natural bone, in which granules of composite nano and micro HA of high open porosity are immersed, which enables high mechanical stability of the scaffold and its exceptional osteoinductivity. The surface micro-fibers are spaced apart, so that they allow an unimpeded flow of nutrients and metabolites of bone tissue decomposition, while the suitable dimensional design and the design of the form of the fillers ensure multimodality of pore size distribution, with the adjustment of their kinetics of dissolution or combustion, the high proliferative ability of such constructs is ensured, which due to their mechanical properties and high bioresorbability are very suitable for the rehabilitation of large bone defects, including defects related to long bones and for their extension.

A brief description of the figures and the presentation of the tables they reflect reflect the essence of the innovation

Fig. 1. CT images of the scaffold granules, which are the structural parts of the implanted bone graft

Fig. 2. Micro CT image of the 3D graft and repaired parts of the rabbit ulna with the microarchitecture of the construct. The total porosity of the graft of about 68% provides suitable conditions for bone growth. The new bone is completely formed in the area of contact of the graft with the intact part of the bone, while in the central part there is a part of the unexploded graft. Within the complete structure of the graft, quality trabecular bone was formed.

Fig. 3. On radiographs of rabbit antebrachocarpal bones taken three weeks after osteotomy and implant placement (left image), signs of formation of calcareous shadows of endosteal callus on the edges of the proximal diaphysis of the ulna, as well as on smaller sequestrations located at the level of the fracture, can be observed. The callus is in the form of fluffy, spotted, milky-white formations that intensify the physiological macrostructure of the bone. The fracture lines are vaguely defined and without distinct traces of bony apposition of new tissue. The shadow of the graft is imbibed with lime shadows and sinks into the surrounding bone structures. There is also a minor soft tissue swelling that blends into the shadow of the adjacent muscles. Nine weeks after surgery (picture in the middle), the fracture line is indistinct due to impregnation with mineral deposit and newly formed bone tissue, which strongly penetrates the construct on both sides where the intact bone is located. The shadow of the construct is less pronounced than in the previous phase of the radiograph and is partially replaced by newly formed bone. The spongiosa of the ulna in the line of discontinuity is condensed and there is noticeable thickening of the compact bone. There was ebrunization of the spongiosa on the middle diaphysis of the radius. On radiographs of rabbit antebrachocarpal bones taken 12 weeks after the completed osteotomy and placement of the bone graft (implant) (right image), signs of intensive formation of endosteal and periosteal callus at the fracture sites can be observed, which in the form of well-formed deposits permeate and coat the bone. The fracture lines are barely visible because they are permeated by newly formed bone tissue that wedges into the graft from the proximal and distal sides. The shadow of the graft is smaller than in the previous phase of imaging and has been replaced to some extent by newly formed bone substance. The spongiosa of the ulna is patchily condensed at the level of the line of interruption of continuity, and a considerable thickening of the compact is also observed. Eburnization of the spongiosa is visible on the middle diaphysis of the radius.

Fig. 4. SEM: Nanotopology of the inner walls of the scaffold

a) Lamellae longer than 5 μm; b) fibers with a length of 5-10 μm and a diameter of 40 nm and c) needles with a length of 1 μm and a diameter of 40 nm

Fig.5.3D (a) and 2D (b) AFM: Channel length 30 μm, width 3 μm, branch thickness: 3-5 μmm. Template driven orientation

Fig. 6. Histological results of an in vivo implanted graft implanted in the ulna of a rabbit, from which a 2.2 cm long section of bone was cut: Toluidine blue staining of a sample of newly formed bone tissue. Enlarged image above. The defect is almost completely filled with newly formed bone tissue. A discrete line of demarcation is noticeable, it represents the boundary between mature and immature new bone. Numerous graft particles are present (red arrows), surrounded by bone tissue, without the presence of soft tissue (10x). Red square: Hyaline cartilage is dominant, which indicates that endochondral ossification occurs in addition to endesmal. Green square: Discrete material particles are surrounded by newly formed bone tissue, which is predominantly of lamellar structure (100k). Yellow square: Morphological patterns of nuclei formed in osteocyte gaps indicate an intensive process of bone reparation. The scaffold particles are completely immersed in the newly formed bone, which indicates excellent osteoconductivity of the bone graft (100 h).

Fig. 7. Goldner trichrome staining of newly formed bone. Enlarged image above. Green color indicates mineralized, while red color indicates unmineralized new bone. Yellow square: Wide area of hyaline cartilage and predominant presence of unmineralized bone tissue indicates active osteogenesis (40x). Green square: Endesmal and endochondral types of ossification are observed. Newly formed bone is mostly immature and unmineralized (100x). Blue square: Particles of material (blue arrows) are completely incorporated into the structure of the newly formed bone tissue, without the presence of fibrous tissues between them and the new bone (100x).

Fig. 8. Critical defect in alveolar bone filled with implanted HA 5, 15 and 25 weeks after implantation

Fig. 9. The compact newly formed bone completely filled the critical defect in the alveolar bone and completely replaced the implanted HA

Table 1. 3D analysis of the architecture of the newly formed bone: All morphometric elements of the 3D architecture of the graft and newly formed bone were analyzed and compared with the data that are characteristic of the intact part of the bone, in the immediate vicinity of the implanted graft, such as the number of layers, their position, volume of bone tissue, surface area of bone tissue, surface intersection, thickness of newly formed bone, factors of trabecular patterns, trabecular separations, moments of inertia, open and total porosity, trabecular thickness, show that the material provided by our invention is in very close agreement with the corresponding factors for natural bone.

Detailed description of the invention

Repair and reconstruction of large bony defects, such as lengthening of the lower extremities, is a serious clinical challenge. The formation of such bone defects can be caused by congenital abnormalities, trauma or disease. This problem is still very current. So far, none of the scientific approaches have shown long-term effectiveness comparable to natural bone. Today's tissue engineering involves stem cells and some appropriately selected scaffold that should enable the most efficient response. Ceramic scaffolds such as macroporous HA have shown some initial clinical success. But, because ceramic materials are brittle, scaffolds are prone to premature breakage. Some, although bioactive, are not resorbable and show a serious risk of infection after a number of years. Many are not resorbed even after 6 years.

A biomechanically stable scaffold with good mechanical induction properties is therefore desirable.

Although bioresorbable polymer scaffolds have been used for a long time, only a few of them have been designed for use in long bone tissues. This is because some of these polymers have a high glass transition temperature (PGA and PLA). In addition, they are brittle and break down too quickly, which can cause long-term problems during the bone remodeling process. In addition, a large proportion of such polymers creates as a by-product of degradation, metabolites that prevent the proper development of cells and bone growth. Another problem is that large grafts of a given species do not have enough channels to provide a rich vascular vasculature for the removal of waste products as well as the delivery of nutrients throughout their volume.

With our invention, the porosity and pore size, which play a key role in bone formation in vivo, is extremely fine-tuned. Thanks to the greater porosity and pore size, it leads to faster bone growth, although the mechanical properties of the scaffold are reduced. The optimization of these two mutually contradictory factors, which was realized in our invention, gives the best efficiency of the scaffold. The subject invention describes the manufacturing process and design of our highly porous scaffold, which has a central channel and multiple side channels, which, despite such optimized porosity, is resistant to breakage, but also has sufficient stability against shearing, torsional and twisting forces. The slow degradation of the scaffold, which is made possible through the optimization of the solubility product of the scaffold, ensures that the 3D matrix maintains its structural integrity and mechanical properties during the complete bone tissue repair process. Scaffolds, which are described in this patent, are suitable for bone regeneration, in various types of bone defects, which include periodontology, maxillofacial surgery, but also more widely as reconstructive parts of bones wherever there is a need to repair defects or to create conditions for new "anti-aging" therapy and applications in the field of plastic surgery and orthopedics. In addition, the scaffolds described here are ideal substitutes for long bone sections in bone tissue engineering. Also, it is possible to influence the increase in the length of the bones in the upper and lower extremities of the human body by using them.

The design of the central channel provided by this approach enables optimal proliferation of cells, bone marrow, growth factors, drugs and their combinations. The outer part of the graft is mainly made of porous bioresorbable polymers or composite nano-HA - biodegradable polymer. The graft has a realistic shape of the part of the bone it replaces. The very process of making the graft can be performed in such a way that it has a very diverse design, which includes sub-elements of different shapes, depending on the pre-desired architecture (elliptical, oval, cylindrical, conical or their combination).

Bioresorbable polymers, natural and synthetic, include polylactides, polyglycolides, polycaprolactones, polyanhydrides, polyamides, polyurethanes, polyesteramides, polyorthoesters, polydioxyanones, polyacetals, polyketals, polycarbonates, polyorthocarbonates, polyphophazenes, polyhydroxybutyrates, polyhydroxyvalerates, polyalkylene oxalates, polyalkylene succinates, poly(maleic acids), poly(amino acids), polyvinylpyrrolidone, polyethylene glycol, polyhydroxycelluloses, chitin, cytosan, , and their copolymers, terpolymers, or other suitable combinations. Suitable bioresorbable polymers are selected based on their mechanical and degradation properties and acceptability for the bone remodeling process. Based on previous experiences, preference in this invention is given to polylactide (PLA), poly(lactide-co-glycolide) and polycaprolactone polymers, because they possess the best combination of degradation and mechanical characteristics,

The architecture of the external part of the graft consists of a series of composite layers composed of nanohydroxyapatite granules and microfibers of bioresorbable polymer (PLA). The printing is done so that the micro-fibers are spaced between 400 and 800 μm, which can be controlled by the software used during printing.

The micro-fibrous graft architecture is designed in layers, so that each supporting layer of microfibers is placed at a different angle to the previous layer. The layers can be placed so that one in relation to the other captures angles from about 0° to about 90°.

The outer part of the graft is wrapped around the inner part. Depending on the expected load of the graft, that part contains 2 or more layers. In that part, the mesh of micro-fibers is made of a porous bioresorbable composite, with an increased proportion of polymer and a slightly smaller proportion of nano-HA, while in the inner part the predominant proportion is nano-HA. Both parts are in very close contact by using a short-term thermal treatment at 90 °C, which conditions better interconnection of the different layers. The thickness of the printed layers is chosen in such a way as to provide the construct with maximum mechanical characteristics and very good proliferative properties. The thickness of the outer layer was usually 1.5-2.5 mm, while the remaining thickness was made up of the inner layer, whose thickness was of the order of 1-2.5 cm. Pore sizes ranged from less than 100 μm to the order of 800 μm, with the largest proportion of pores in the 150-300 μm range.

In addition, scaffolds should have a certain mechanical strength. The way in which the layering is done in the outer part of the graft and the choice of the polymer composite itself determines its mechanics, which in our cases amounted to 5-50 MPa. The granules themselves, which make up the basic part of the inner part of the graft, had a strength of around 5-15 MPa. The values of the outer part of the graft, depending on the number of layers, are also designed for higher values, which can range up to 100 MPa, if necessary due to the load that the bone suffers in vivo. The stated values were sufficient to obtain satisfactory mechanics of the bone under load, which is extremely important for the stability of the graft in the process of its transformation into natural bone.

The inner part of the graft forms a central channel, the diameter of which is suitable for the transport of nutrients, metabolites, cells, growth factors and various combinations thereof. The central channel has a diameter that is suitable to perform all the functions of it, especially the vascular function and the harmonious flow of cells, growth factors and nutrients to all parts of the graft, following the rules of fluid dynamics. Its geometry is defined based on the diameter and canal architecture of the natural bone, which is replaced by the given graft. Its typical diameter is about 5-10 mm.

The graft may have lateral or secondary canals. Such channels enable the connection of blood vessels. They can be randomly and uniformly distributed. In one embodiment, the side channels are evenly spaced. The spacing is chosen to satisfy all the requirements related to the even flow of all the above-mentioned elements, especially progenitor cells and growth factors, as well as metabolites, throughout the entire volume of the graft, which are expected from such a graft. Their diameter is usually around 250 μm, while their maximum dimensions can have a diameter of up to 700 μm. The smallest dimensions of the diameter of these channels are about 50-100 μm.

The graft generally has two ends. The ends can be open or closed, or open at one end and closed at the other. One or both ends of the graft can be closed by adding one or more layers of micro-fiber mesh. Microfibers in the outer and inner part of the graft can be the same or different.

The scaffolds/grafts described here are especially used in bone regeneration, in the repair or filling of bone defects, in oral maxillofacial surgery. In addition, such grafts are ideal for tissue engineering of long bones. They make it possible to increase the length of the bones of the upper and lower limbs of the human body. The graft can be implanted with or without the addition of bioactive and/or inert agents. In addition, the grafts can be cultured in vitro with cells isolated from a donor, such as the patient, before implanting the graft into the patient, to accelerate osseointegration.

after implantation in the patient.

Examples

Bone tissue regeneration using PLA - nano-HA bone graft in rabbit ulna

Examples of nanoHA synthesis

NanoHA powder was prepared by the method of hydrothermal synthesis, with the use of copolymers of polyethylene vinyl acetate and polyethylene versatate, using pressures of 5, 25 and 50 bars, and temperatures of 125, 150 and 180 C, for a period of 8 to 24 hours. Based on the given values of the synthesis parameters, an experiment matrix was created, in which each synthesis parameter was crossed with each other, on the basis of which a variety of powders of different particle sizes, surface-to-volume ratio and crystallite size, as the basic structural elements of the given nanoHA powders, and therefore powders with different chemical solubility, measured over the solubility period of each of the obtained powders, were obtained. The resulting powders had a rod-like to hexagonal shape, with dimensions from 50 to 200 nm. After that, the powders were subjected to a mechanochemical process of particle size reduction to dimensions of around 10 nm, in order to obtain even greater chemical and biological activity of the obtained powder.

Additional design of nanoHA during the hydrothermal synthesis process was carried out by partial replacement of Ca<2+> ions with Mg<2+>, Zn<2+>, Sr<2+> ions and PO43- ions with CO3<2-> and SiO4<2-> ions, within the limits of 0.5-3.5 at%, after which the system was again subjected to the dissolution process in a solution with optimally defined pH values, in order to determine the parameters of its degradation rate and adjust to the optimal value.

nanoHA, which showed an ideally adjusted rate of chemical degradation, was used for the preparation of granules and grafts of the nanoHA polylactide-glycolide composite system.

Graft preparation

The graft was prepared by using a composite of PLA and nano-HA powder for printing the outer part of the graft in a ratio of 80:20 and nano-HA granules coated with a thin film of PLGA for printing the inner parts of the graft, with a dominant share of nano-HA granules. HA granules were obtained by using a multi-stage template consisting of nano-HA whose solubility was previously adjusted by the addition of appropriate Mg or Zn ions, as well as silicate ions, to the solubility value of natural bone. The microporous template was provided by the use of hydroxymethyl cellulose and cellulose ethers, whose molecular weight varied between 30,000 and 100,000 Da, with the addition of a small amount of activated montimolyronites, in a suitable concentration, which ensures good rheology of the system and the adjustment of the size of micro and medium-sized pores, while a polyurethane mesh, with a mesh size of about 1-1.5 mm, was used as a macrotemplate, in thin slices, which were suitable for the technique of impregnation of such a structure with a given suspension. Nano-HA also served as a reinforcing component. After drying, the given system was thermally treated at 1100 °C for better sintering. A similar result was obtained through the process of leaching the hydrophobic polymer part of the template using a certain multimodal distribution of polystyrene, and subsequent sintering at temperatures of about 1100 °C. Different porosities are obtained at different ratios of polyurethane network-based templates and suspensions with nano-HA. The porosities obtained during the experiments were between 60 and 78%. For biocompatibility and implantation tests, samples with porosity of 70-75% were used.

Nanopowder of activated HA was used for the design of the outer network of the graft, while HA granules pre-impregnated with PLGA were used for the inner part of the graft. Through an adequate printing method, which was described in detail in the previous part of this invention, grafts were obtained that can be used as reparative elements for long bones as well. The results obtained during numerous biological tests showed that the obtained constructs can be successfully used for bone tissue repair not only in maxillofacial and oral surgery, but also for the extension of limb bone tissue, reconstructive surgery, when replacing parts of bone affected by cancer, etc.

Three-dimensional (3D) modeling and printing of grafts

Computed tomography (CT) was used to scan a rabbit ulna, which was obtained preoperatively, from an anesthetized animal, using a Scanora 3D (Tuusula, Finland) 3D scanner and the following parameters: 13 mA, 90 kV, and a voxel resolution of 0.2 mm. Subsequently, the CT images were converted into a 3D model using Slicer software, version 4.3.1 (Brigham and Womans Hospital, Inc, Boston, MA) and Blender software. Next, the 3D model was imported into Autodesc 3D Max 2010 (Autodesc Inc.). The defect dimensions and borders (22 mm X 7 mm) were pre-planned and an identical graft was printed based on it. This was verified using Meshmiker software and imported into Slice3R software (Affero Inc.) for G-code generation. A printer made according to our own design (Albos doo, Belgrade, Serbia) and Pronterface software were used to print the 3D bone construct, using the following parameters: printing speed 50 mm/s, layer height 0.2 mm, infill of 60%, 0 shell thickness and no material support. The scaffold was produced by combining a biodegradable PLA and nano-HA composite with PLGA, with a gradually increased percentage of nano-HA towards the central part of the construct.

Microporosity test of printed graft and newly formed bone

Each bone sample was scanned in the dry state at a resolution of 10 μm using micro-CT (SkyScan 1172 x-Ray Microtomography, SkyScan, Kontich, Belgium). Acquisitions were performed at a voltage of 85 kV, a tube current of 118 μA, an exposure time of 1000 ms, using an aluminum and copper filter with a thickness of 0.5 mm, and a rotation of 180°. The obtained recordings were reconstructed using NRecon v.

1.6.9.8 software with beam correction of 45%, ring artifact correction of 6%, and attenuation of 2. Images were then analyzed using CTAn 1.16.4.1 software.

The following parameters of the microarchitecture of the cortical implant or newly formed bone were examined: cortical thickness (Ct.Th, mm), which represents the average thickness of the formed cortical bone; porosity (Ct.Po, %), which represents the pore volume in relation to the total volume of the implant or newly formed bone, pore diameter (Po.Dm, μm) which represents the average pore diameter and pore interconnection density factor (Po.Sp, 1/mm<3>), which represents the average distance between pores.

In vivo implantation of a personalized graft in the rabbit ulna

A rabbit previously scanned for graft design was subjected to intramuscular premedication (combination of ketamidor (ketamine hydro-chloride) 10%, Richter Pharma Ag -Austria, 35 mg/kg, with xylazine (Xilased) 2%. Bioveta, Czech Republic, 5 mg/kg). Butorphanol was used for analgesia in an intramuscular dose of 0.1 mg/kg (Richter Pharma Ag, Austria). A linear skin incision of 3.5 cm was made in the ulna area under local anesthesia (lidocaine chloride 2%, Galenika ad Srbija). The soft tissues were lateralized, and a 3D printed bone construct that matched the shape and dimensions of the defect cut from the bone was implanted into the ulna. Namely, the defect was created using a known distance from the proximal part of the ulna (30 mm), which enabled precise positioning of the graft. After that, an osteotome section was made through the entire thickness of the bone (diameter 22 mm). The surgical wound was closed at multiple tissue levels with surgical sutures (Vicryl, ETHICON, 3-0). Butorphanol was used every 4 hours to reduce pain 5 days after surgery. The rabbit was sacrificed 12 weeks after implantation. After that, the ulna bone with the implanted construct and 10 mm of surrounding old bone with the bony defect region were removed and fixed in 4% neutral formalin, for histological analysis that followed.

Evaluation of the obtained results

2D radiography: Antero-posterior (AP) and lateral radiography using X-ray analysis after 12 weeks

3D micro CT evaluation: Skyscan 1072, voltage 85 kV and current 118 μA. Trabecular thickness, trabecular separation and BVF (bone volume fraction) were determined after 12 weeks.

Bone mineralized density (BMD): Lunar PIXImus densitometry, using a machine to scan a region of the newly formed bone tissue sample after 12 weeks, with the scanned sections of the sample divided into 0.08 cm squares.

Histological and histomorphometric analysis after 12 weeks

Specimens for histological analysis were prepared by a standard procedure that included fixation in 4% buffered formaldehyde, decalcification in formic acid, dehydration, and embedding in Paraplast. Longitudinal sections with a thickness of 4 μm were stained with hematoxylin and eosin, the Goldner trichrome technique, which is suitable for the presentation of bone tissue, and Toluidine blue, which is suitable for assessing bone vitality. Two-dimensional photographs were made with digital o oapara om on , ema a . s o o a n a z a s s t r a n s of an omni-axis microscope (Leitz Rada Lux M Fuorescence Microscopy, Ernst Leitz Weclar GmbH, Germany).

Histomorphometric analysis was performed using a software package (Nikon University Suite, version 4.3, Leica Microsystems, Germany). Four central sections were analyzed per sample, with 50 µM spacing between sections.

The morphometric assessment was performed using a final magnification of 400x, and the following parameters were analyzed:

1. Presence, total surface and histological characteristics of newly formed bone tissue. These are also the most important parameters from the point of view of bone regeneration.

2. Total surface area of mineralized and non-mineralized bone. The Goldner trichrome staining method was chosen for this analysis because it allowed mineralized bone to be shown in the green part of the spectrum and unmineralized bone in the red part of the spectrum. The presence of unmineralized bone is the most important parameter in the assessment of active osteogenesis and the efficiency of the bone remodeling process.

3. The total surface area of the newly formed bone marrow.

4. Total surface area of graft and connective tissue particles.

5. The presence and number of inflammatory infiltrate cells, which is the most important parameter of the biocompatibility of the tested materials in vivo. Based on the assumption that the efficiency of regeneration will differ in the central region of the repaired bone compared to its peripheral parts that are in contact with the part of the intact bone, all histological and histomorphometric parameters were determined in both of these areas.

Immunohistochemical staining for confocal microscopy

Formalin-fixed and Paraplast-embedded rabbit ulna bone tissue sections were prepared in the form of thin slices, air-dried and deparaffinized in a series of alcohol mixtures (2x5 minutes in xylene, absolute ethanol, 96%, 70% ethanol and distilled water). Sections were stored for 25 minutes at 90 °C in antigen unmasking solution (Vector Laboratories, USA). To block nonspecific staining, sections were incubated in 5% bovine serum albumin (BSA, Sigma-Aldrich) diluted in PBS (PBS-BSA) for 30 min before the addition of primary and secondary antibodies. Purified primary antibodies to osteopontin, BMP2, and osteocalcin (Thermo Fisher Scientific; dilutions 1:200, 1:100, and 1:100 in 5% BSA) were applied to the samples for 24 h at 4 °C, followed by 1 h incubation with a second Alek Fluor 488-conjugated antibody (Thermo Fisher Scientific, dilution 1:500 in PBS) in humid dark chamber. For nuclear staining, propidium iodide (Thermo Fisher Scientific concentration 1 µg/ml) in PBS was added at 5 min intervals at room temperature in a dark humidified chamber. Each step was followed by 3 washes in PBS for 5 min. Finally, the examined parts were covered with fluorescent medium (DAKO, Glostrup, Denmark) and examined with a confocal microscope (Laser Scanning Microscope, LSM META 510, Carl Zeiss, USA). Sections were examined using 20 or 40 objectives. To confirm the specificity of the staining, a secondary antibody staining control was performed (eg the primary antibody was replaced with 5% BSA).

Results

The results of the histological analysis showed that the healing of the defect went smoothly, without complications in terms of necrosis, infection or bleeding. After the twelfth week of healing, the surgical defect was filled with newly formed bone tissue and remnants of still unabsorbed graft particles. The newly formed bone tissue is in continuity with the "old" bone, separated only by a very discrete line of demarcation. In this region, as well as in the newly formed bone, small, individual graft particles were observed, which the bone tissue completely surrounded, without interposing connective tissue, which speaks in favor of strong osteoconductive properties of the material. Phlistological analysis showed that a small part of the newly formed bone was mineralized, while the largest part was still unmineralized, which speaks in favor of active osteogenesis. Also, the newly formed bone tissue mostly showed signs of lamellar, mature organization, with vital osteocytes, concentrically placed around the Haversian canals, but fibrous, immature bone was also present, which speaks in favor of active bone tissue remodeling. It was observed that the bone tissue largely shows a spongy structure with newly formed bone marrow, between the trabeculae. Parts of hyaline cartilage were also observed, which suggests that both methods of ossification, direct and indirect, are present.

The results of the histomorphometric analysis show that the total area of the newly formed bone is 37.91%, and the newly formed bone marrow, located in the cavities of the trabecular bone, is 40.41%, which in total makes up almost 80% of the surgical defect. This finding speaks in favor of almost complete bone regeneration. Such a favorable therapeutic result can be related to the extraordinary osteoconductive properties of the material, the size of the particles, their porosity, but also the ideal timing between osteogenesis on the one hand and resorption of the material on the other. Graft particles made up 21.16% of the defect surface, and most of them were embedded in the newly formed bone tissue. Connective tissue was only around the periphery of the defect. No inflammatory infiltrate cells were observed in it, which speaks in favor of the material's biocompatibility.

The essence of the technical part of the solution is found in the formulation of a completely new procedure/process for obtaining a specific ceramic-polymer compact, which is mostly contained in the realization of a specific geometry, which in its hierarchy contains interconnected macrostructural elements of the order of hundreds of micrometers and elements of the lowest level of the hierarchy of the order of only a few nanometers. Such geometry, with its specific features, enables the simple proliferation of bone cells in all directions, which, due to the extremely well-defined morphology of the internal walls at the micro and nano level and active polymer structures, very easily stick and adhere to the walls of the scaffold, which makes their signaling and information transfer extremely efficient.

This made it possible, first of all, to obtain carbonate HA powder with a shifted stoichiometry of a well-defined shape and particle size distribution at the nanolevel using the hydrothermal method, combined with a surface-active copolymer of polyethylene vinyl acetate and polyvinyl aversatate. The final nanometer design of the powder was given using the mechanochemical method. The second stage of the process included obtaining the scaffold itself (a ceramic support with a well-defined distribution of pores and morphology of the internal walls, using the polymer model method to define the form of the scaffold and the sintering method to achieve its final mechanical characteristics, while the last stage of the process involves the deposition of very thin polymer films (most often based on PLGA) and biomimetic treatment to finalize the design of the surface of the support, by inducing biological apatite on the surface of the given ceramic support-polymer structure. Process parameters in all stages are defined very precisely, which makes the process highly reproducible.

Good chemical characteristics of the scaffold imply, first of all, the optimal rate of degradation of the scaffold, which should be in dynamic balance with the rate of formation of new bone through the mediation of osteoblast bone cells. In order to achieve such scaffold characteristics, HA powders are designed so that their chemical degradation rate matches the chemical degradation rate of biological HA. These systems have a shifted chemical composition in relation to the stoichiometric composition of apatite with embedded ions, most often Mg<2+> and Zn<2+> cations and CO3<2->, SiO4<2-> anions in positions corresponding to calcium ions and phosphate ions. The rate of degradation was investigated through the degradation program of the given powders in the form of capsules with a thickness of 1 mm with the use of trisbuffer and HEPES as reagents to stabilize the pH value of the solution. Follow-ups were performed in long time intervals, within the established time scale to define the degradation kinetics of the hydroxyapatite system and for each of them to determine the appropriate solubility product.

In order to fully demonstrate the practical effect of the obtained bone granules and bone graft, they were subjected to a wide range of biological research, which, in addition to implantation tests, included a series of complementary tests that include a cytotoxicity test, genotoxicity (comet test), LDH test, alkaline phosphatase test, on L929 fibroblast cell cultures, as well as sensitivity tests (cutaneous and subcutaneous irritation), as well as acute and subchronic toxicity tests, and a series of tests performed on stem cells, which showed the efficiency and speed of the differentiation process of stem cells into osteoblasts, on the surface of the given constructs. All tests showed the material's excellent osteoinductivity and sensory integrability, with exceptional bioacceptability and negligible cytotoxicity, which the material recommends for clinical application tests in various areas of bone tissue implantology.

Claims (6)

The patent claim relates to: 1. An osteoinductive graft based on nano-HA and a copolymer of lactic and glycolic acid, indicated by this, refers to the design of a bioresorbable osteoinductive graft, by the method of extrusion printing, based on a composite consisting of activated nano-HA, covered with a thin film of PLGA, and PLA, at different ratios of the two components inside the central part of the graft and on its outer sheath. 2. Osteoinductive graft based on nano-HA and copolymer of lactic and glycolic acid according to claim 1, indicated that it refers to the chemical process of synthesis of nanohydroxyapatite, by a combination of hydrothermal method and mechanochemical method, under pressures of 5-30 bar, at a temperature of 125-180 °C, during a time of 8-24h, with the presence of surface-active substances of copolymers of polyvinyl acetate and polyvinyl vesatate. 3. Osteoinductive graft based on nano-HA and copolymer of lactic and glycolic acid according to claim 1, indicated that it refers to the solubility optimization process, in accordance with the procedure for obtaining nanoHA described in claim 2, which involves the incorporation of various ions such as Mg<2+>, Sr<2+>, Mn<2+> and Zn<2+>, or SiO4<2-> ions and their combinations, into the crystal lattice of HA, using appropriate chemicals that are carriers of such ions, in concentration limits between 0.5 and 3.5 at% in relation to the corresponding ions inside the nano-HA, which they exchange (Ca<2+> and PO4<3-> ions). 4. Osteoinductive graft based on nano-HA and copolymer of lactic and lactic acid according to claim 1, indicated by that it refers to nano-HA spherical or rod-shaped forms with dimensions of up to 100 nm in length and a diameter of several nanometers, obtained by hyprothermal synthesis procedures with the application of surfactants based on polyethylene vinyl acetate and polyethylene vinyl versatate, as well as other related synthesis methods, such as the method of combined agents, the method of double micelles and methods syntheses in a periodic ultrasonic or radiofrequency field. 5. Osteoinductive graft based on nano-HA and copolymer of lactic and glycolic acid, indicated by this, refers to granules, obtained by applying a polyurethane network as a macro template and a nano-HA network in a suspension of hydroxymethyl cellulose (HC), which forms a thin film on the surface of nano-HA as a micro template and particles of the dispersing phase of the order of 100 to 300 μm in size, with the addition of highly adhesive starch ethers, montymorillonite and hexa of metaphosphates, which ensure that the system does not mechanically collapse during rapid sintering and enable the strengthening of the bonds between the nano-HA particles, creating highly porous compacts with satisfactory mechanical characteristics. 6. Osteoinductive graft based on nano-HA and copolymer of lactic and glycolic acid, indicated by this, refers to the method of printing the graft, according to which each subsequent row is placed at an angle of usually 50° in relation to the previous layer, thus producing interconnected pores and ensuring good mechanical characteristics of the graft and its application not only in supercritical defects of all kinds in maxillofacial surgery, but also in loaded parts of the bone, as well as in lengthening the length of the bones of the upper and lower extremities, and solving long bone defects and large-scale incisions, as well as in the field of reconstructive surgery of bone tissues that were previously affected by bone cancer or when applied in the field of plastic surgery for reconstructive procedures on facial bones. 20