FIELD OF INVENTION

The present invention related to a dual density hydroxyapatite scaffold and a process of preparing thereof. More specifically, the present invention is related to a scaffold to be used to connect ends of broken or damaged long bones in mammals such as humans.

BACKGROUND OF INVENTION

Tibia bone diaphysis is the most common site for a bone defect due to poor soft tissue coverage especially at the anteromedial site. The goal standard treatment for a bone defect would be placement of autologous bone graft. However, this technique causes morbidity in terms of pain and haemorrhage at the operation site. Apart from that, there would be risk of infection as well as non- union. Graft failure due to insufficient graft vasculature would result in decreased mechanical stability. Alternatively, vascularized auto graft can be used, but it is more technically demanding as well as time consuming.

Allograft and xenograft poses high incidence of graft rejection. In addition, maintenance of bone banks and operating procedure involves considerable expenses. The conventional method, "The Iilizarov technique", based upon the principle of distraction osteogenesis has shown good outcome in addressing bone defects. Nevertheless, it involves long duration of time and risk of pin tract infection, thus stated as inconvenient for patients.

Recent research efforts focus on bone graft substitute involving growth factors with a naturally derived or synthetically manufactured, mechanically supporting scaffold. Holding to this idea in mind, our aim in this study is to provide a suitable scaffold and a bone growth stimulating agent that results in comparable or even better bone healing compared to the gold standard tricortical autograft transplantation. SUMMARY OF INVENTION

Accordingly, the present invention provides a dual density scaffold used to connect ends of broken or damaged long bones in mammals such as humans comprising an inner porous layer, an outer dense layer, at least one bioactive agent, at least one inert agent, or a combination thereof, wherein the outer dense layer having a particle size of <200 nM, wherein (a) the inner porous layer is made of a ceramic slurry containing 60% beta-tricalcium phosphate and 40% hydroxyapatite and (b) the outer dense layer is made from 100% hydroxyapatite wherein the scaffold having ends that are configured to act as sleeves and fit to respective end of the broken or damaged long bones.

Furthermore the present invention also provides a method of fabricating the above-mentioned dual density scaffold wherein the method includes the steps of (a) preparing of mould according to a 3 -dimentiona l computerised topography (3-D CT) scan data, (b) preparing ceramic slurry containing 60% of hydroxyapatite and 40% of beta-tricalcium phosphate to produce the inner porous layer and 100% of hydroxyapatite to produce the dense outer layer with a particle size of <200 nM, (c) casting of the ceramic slurry in the mould, (d) creating the inner porous layer by adding porogen such as sodium chloride or polymeric sponges soaked in the ceramic slurry containing monomers and cross-linkers, (e) sintering of outer dense layer and the inner porous layer to form the scaffold, (f) sterilizing the scaffold, (g) harvesting blood and cells from a mammal patient, (h) culturing of cells from the patient, (i) isolating platelet-rich plasma from the blood of the patient, (j) mixing of the cells and platelet-rich piasma and CaCl ₂ to create a cell suspension, (k) seeding the cell suspension into the inner porous layer to form a cell-seeded scaffold, (1) immersing the cell-seeded scaffold in a culture media and (m) incubating the cell- seeded scaffold prior to implanting the scaffold into the patient BRIEF DESCRIPTION OF THE DRAWING

Figure 1 is a photograph of the dual density scaffold having the inner porous layer and outer- dense layer according to the preferred embodiments of the present invention.

Figure 2 is a flow chart describing the method of fabricating the dual density scaffold of Figure

DETAILED DESCRIPTION OF THE PREFERED EMBODIMENTS

The present, disclosure may be best understood with reference to the detailed figures and description set forth herein. Various embodiments are discussed below with reference to the figures, However, those skilled in the art will readily appreciate that the detailed descriptions given herein with respect to the figures are simply for explanatory purposes as the methods and systems may extend beyond the described embodiments. For example, the teachings presented and the needs of a particular application may yield multiple alternative and suitable approaches to implement the functionality of any detail described herein, Therefore _. , any approach may extend beyond the particular implementation choices in the following embodiments described and shown.

References to "one embodiment," "at least one embodiment," "an embodiment," "one example," "an example," "for example," and so on indicate that the embodiment(s) or example(s) may include a particular feature, structure, characteristic, property, element, or limitation but that not every embodiment or example necessarily includes that particular feature, structure, characteristic, property, element, or limitation. Further, repeated use of the phrase "in an embodiment" does not necessarily refer to the same embodiment.

The present invention will now be described in reference to Figure 1. The scaffold comprises an inner porous layer, an outer dense layer, at least one bioactive agent, at least one inert agent, or a combination thereof, wherein the outer dense layer having a particle size of <200 nM, wherein (a) the inner porous layer is made of a ceramic slurry containing 60% beta-trieaicium phosphate and 40% hydroxyapatite and (b) the outer dense layer is made from 100% hydroxyapatite wherein the scaffold having ends that are configured to act as sleeves and fit to respective end of the broken or damaged long bones. The inner porous layer comprising micropores having an average diameter of 250 μΜ to 500 μΜ and a percentage porosity of 40% - 60% in order to facilitate cell penetration and vascularization with an optimal percentage porosity of 50%. The outer dense layer comprising micropores of an average diameter of less than 5 μΜ and a percentage porosity of 5% - 10% in order to achieve a mechanical strength and stability to act as a sleeve bridging ends of the segmental bones. The inner porous layer has a compressive strength of 5-35 MPa while the outer dense layer has a compressive strength of at least 115 MPa. The outer dense layer has a radial thickness ranging from 2 mm - 5 mm. The inner porous layer occupies at least two thirds of the scaffold. The at least one bioactive agent is osteoprogenitor cells or osteodifferentiated stem cells and platelet rich plasma or plasma derived fibrinogen. The at least one inert agent is a polymerizing agent such as calcium chloride (CaCl ₂ ).

The present invention will now be described in reference to Figure 2, A method of fabricating the dual density scaffold wherein the method includes the steps of (a) preparing of mould according to a 3-dimentionai computerised topography (3-D CT) scan data (bj the preparation of ceramic slurry containing 60% of hydroxyapatite and 40% of beta-tricalcium phosphate to produce the inner porous layer and 100% of hydroxyapatite to produce the dense outer layer with a particle size of <200 nM, (c) casting of the ceramic slurry in the mould (d) creating the inner porous layer by adding porogen such as sodium chloride or polymeric sponges soaked in the ceramic slurry containing monomers and cross-linkers, (e) sintering of outer dense layer and the inner porous layer to form the scaffold (f) sterilizing the scaffold (g) harvesting blood and cells from a mammal, patient (h) culturing of cells from the patient (i) isolating platelet-rich plasma from the blood of the patient (j) mixing of the cells and platelet- rich plasma and CaCl ₂ to create a cell suspension (k) seeding the cell suspension into the inner porous layer to form a cell-seeded scaffold (1) immersing the cell-seeded scaffold in a culture media; and (m) incubating the cell-seeded scaffold prior to implanting the scaffold into the patient. The following section describes the experimental procedures which are not in reference to Figure 2, Autologous plasma preparation

Fresh sheep blood of 20 millilitres was collected through a venous puncture. Blood was collected in sodium citrate tubes followed by rapid inversion of the tube to deter blood clotting and was kept at ambient temperature during transport to the cell culture facility. It was then subjected to centrifugation at 5000 rpm for 5 minutes. The plasma layer was gently transferred to a new tube with a pipette without disturbing the bottom layer. Centrifugation was repeated and the plasma layer was transferred to ensure complete removal red blood cells.

Sheep bone marrow harvesting

Sheep was anesthetized with Ketamine 1mg/kg bodyweight via intravenous injection. Right iliac crest region of the sheep was shaved, scrubbed, cleaned and covered with sterile drape. One-centimetre skin incision was made over the iliac crest. Five-ten millilitres of bone marrow was harvested using a 50ml syringe via a Jamshidi needle from the sheep iliac crests. The bone marrow was kept in EDTA vacutainers and transferred to the cell culture facility within 24 hours at ambient temperature. in vitro cell culture

Aspirated bone marrow was first diluted with standard culture medium supplemented with 10% foetal bovine serum. Then, mononuclear cells were isolated from the diluted bone marrow via gradient centrifugation over a Ficoll-Paque layer at 5000rpm for 20 minutes and subsequently washed twice with phosphate-buffered saline. Cells were resuspended in culture medium (F12: DM EM 50:50 supplemented with 10% fetal bovine serum) and plated onto a 25cm ² culture flask. All cultures were incubated at 37°C in a humidified atmosphere of 5% COz. Fresh medium was added on the third day. Medium was changed upon substantial cell attachment and later, twice a week. Upon cell confluence, cells were detached by the addition of 0.05% of trypsin-EDTA solution and counted using trypan blue dye-exclusion-method and a haemocytometer. Cells were subsequently sub-cultured at. a standard density of 5000 cells/cm ² . Sub-clituring was performed for 3 to 5 times in order to expand the cells to approximately 30 million. One week before the implantation date, cells were maintained in Osteogenic Medium (culture media supplemented with 10-7 M dexametbasone, 0.05 mg/ml ascorbate-2- phosphate, 10 mM b-glycerophosphate) for one week to induce osteogenic differentiation.

In vitro tissue engineered bone construct preparation

TCP/HA cylinders were firstly pre-wetted with medium. Approximately 30-50 million osteoprogenitor cells were seeded on each cylinder. Cells were premixed with plasma at the ratio of 1 x 106 cells to 100 μL plasma and dropped using a pipette onto the pre-wetted granules. Polymerization of the fibrinogen in the plasma was initiated by the addition of 100 μl of 0.5M CaCl ₂ - The conversion of fibrinogen into fibrin will rrap the cells within the scaffold. The cell-seeded scaffold (bone constructs) will then be immer sed in osteogenic medium for one Vv'eek in a CO ₂ incubator to induce osteogenic differentiation. After 1 week, the cell-seeded scaffold will then be transported immersed in the osteogenic medium at 4-18°C in a cold box to the operating theatre for implantation.

Animal Surgery

A venous access line will be installed at the right front Sower limb under aseptic conditions, and Diazepam 0.3-0.5 mg/kg BW and Ketamine 3-5mg/kg BW will be injected to induce general anaesthesia. The respective animal will then be intubated with a 9-10 mm silicon endotracheal tube and connected to an automatic respirator (Campbell anaesthetic ventilator) for assisted ventilation with 2L Oz/min, For prophylactic antibiotics, fV Amoxycillin 15mg / kg BW will be given along with analgesia - IM Meloxicam 2 mg/kg BW daily and 1M tramal 2 mg/kg. The animal's heart rate, oxygen saturation and end-tidal carbon dioxide levels were monitored and recorded continuously. Animals (the sheep, average weight: 42.5 kg, age: 6-7 years) positioned right lateral recumbency. The left hindlimb will be shaved and thoroughly disinfected with 0.5% chlorhexidine solution red in 70% ethanol. The animal torso and surroundings were then covered with sterile sheets, the surgical area additionally with Opsite. Autologous, tricortical bone graft harvest from the left iliac crest. The surgical area was shave and disinfected with 0.5% chlorhexidine red in 70% ethanol, A 5-cm incision was made following the iliac crest the inserting musculature was carefully detached and the cortical bone of the lateral os ileum was fenestrated (2 x 2 cm) using a hammer and osteotome. Care was taken not to fracture the ala ossis ilii. The resulting lid was carefully removed with a raspatory and tricortical hone harvest utilizing a bone curette. The lid will be reinserted, and the musculature reattach with 2-0 Vicryl sutures, and the wound dosed in layers. The closed wound was sprayed with Opsite.

4 Dunham pins 5mm will inserted over the tibia (2 proximal and 2 distal). Rods will be inserted and tightened. The right, tibia exposed by a longitudinal incision of approximately 5 cm length on the medial aspect of the limb. Next, the soft tissue inserting to the bone in the designated defect area detach and a wet compress placed between bone and posterolateral soft tissue to avoid damage to proximate nerve and blood vessels during osteotomy. Parallel osteotomies perpendicular to the bone's longitudinal axis perform with an oscillating saw (Stryker) under constant irrigation with saline solution to prevent heat induced osteonecrosis whilst, the bone segment of 3 cm length was excised. Care was taken to completely remove the periosteum within the defect area and 1.5 cm proximally and distally of the osteotomy lines. The iliac bone fragments will be place realign. The wound was closed in layers with a 2-0 Monocryi and a 3-0 Novafil suture for the skin. The closed wound was sprayed with Opsite, covered with pads and bandaged. After recovery from anaesthesia, animals were allowed unrestricted weight bearing.