WO2021205306A1

WO2021205306A1 - Process for obtaining spongy material for bone regeneration

Info

Publication number: WO2021205306A1
Application number: PCT/IB2021/052776
Authority: WO
Inventors: Francesca BOCCAFOSCHI; Marta CALVO CATOIRA; Luca Fusaro
Original assignee: Tissuegraft S.R.L.; Passalacqua Escavazioni S.R.L.
Priority date: 2020-04-09
Filing date: 2021-04-02
Publication date: 2021-10-14
Also published as: IT202000007558A1

Abstract

Process for obtaining a spongy material for bone regeneration, said process providing: - for the recovery of pericardia of bovine origin which are decellularized, lyophilized, reduced into a powder, said powder being enzymatically treated in order to carry the proteins thereof in aqueous suspension form; - that the aqueous suspension, brought to physiological pH and temperature, becomes a hydrogel, with viscosity greater than that of the starting suspension; - that in turn, the hydrogels are subsequently lyophilized, so as to obtain dry and spongy structures.

Description

“Process for obtaining spongy material for bone regeneration”

Description Field of the art

The present description refers to the medical field. More in detail the present description refers to a particular process for obtaining an equally particular material in sponge form to be used in the field of bone regeneration. Prior art

In the context of bone regeneration, bone grafting is widely used and is represented by 500,000 cases per year in the United States alone and about 2,200,000 worldwide. The medical fields most relevant for grafting are orthopedics, neurosurgery and dentistry. 90% of the causes of bone atrophies and defects of the maxillary bones are represented by edentulous and periodontal diseases.

The gold standard is represented by the autologous bone graft which nevertheless does not lack disadvantages including: long duration and complexity of the surgical procedure, cosmetic disadvantages and residual pain in the drawing site. The greatest complication is given by a possible failure of the procedure in case of lack of survival of the osteogenic cells during the transplant; exclusion criteria are also referred to patients of pediatric age or advanced age or in the case of malign pathologies. Other complications have an appearance rate comprised between 8.5% and 20% including: formation of a hematoma, blood loss, damage to components of the nervous system, infections, artery damage and chronic pain at the donor site. As secondary option with respect to the autologous bone graft, the homologous bone graft is widely used in the United States (it represents 30% of the bone grafts and it is available in various commercial compositions such as dowels, chips, strips of cortical or spongy bone. Such therapy consists of using bone drawn from a donor compatible with the patient. Of course, this type of bone has limitations with respect to autologous bone, as well as the possibility of transmission of pathologies if not suitably treated before the implant in the receiving site. The manipulation of the donated material provides for the sterilization (with ethylene oxide or gamma rays) and the freezing or freezing/drying. The main object is to prevent infective complications, but it comes to modify the characteristics that the material initially possesses, reason for which the frozen or frozen/dried bone loses osteoinduction capacity; untreated fresh bone indeed cannot be used since it would create an immune response or it would lead to the transmission of infective pathologies. The frozen/dried bone thus obtained has lower capacity to induce an immune response but is less osteoinductive and has worsened mechanical characteristics than the bone treated with only freezing. As advantages, one notes the lack of complications connected to drawing from the donor site of the patient, while as disadvantages one observes an osteoconduction and a limited osteoinduction and a lack of osteosynthetic capacity.

As improved treatment of the autologous transplant, there is the osteoplatelet gel that consists of the addition of PRP with autologous bone fragments which come to fill the zones where the bone is not present. Even if the use of PRP facilitates the manipulation and the positioning of the bone grafts and accelerates the healing of the soft and hard tissues, the bibliography does not demonstrate a statistically significant advantage in bone regeneration or an evident clinical benefit such to support the use. Other drawbacks of such therapy are its high cost and the time used in the application of the same (Nikolidakis and Jansen, 2008). Alternatively, guided bone regeneration is present (GBR) and it is one of the surgical techniques developed over the last two decades for regenerating the bone and thus allowing the insertion of implants in compromised sites. It is a surgical procedure that consists of physically positioning an impermeable membrane at the cells between the connective tissue and the alveolar bone defect. This barrier prevents the migration of the soft tissues in the defect and creates a protected space in which the coagulation and the graft are stabilized. The migration of epithelial and connective cells is prevented, while the slow proliferation of the osteogenic cells can occur with consequent formation of new bone. The most common complication of tins treatinent regards the early exposure of the membrane in the oral cavity, with its sequence. However, other complications have also been reported, including the formation of an abscess and vascular-nervous lesions, which seek to find a solution by employing membranes made of expanded polytetrafluoroethylene (e-PTFE) due to their proven effectiveness and to the ease of use.

However, new experimental approaches use natural materials such as gelatin (Hamai et al., 2020) for creating lyophilized scaffolds with the addition of inorganic compounds such as hydroxyapatite or octacalcium phosphate, employed in bone regeneration. Several of them employ the platelet-rich plasma added to the scaffold in order to improve the performances thereof (Rodriguez et al., 2013). Frank et al. offer a complete vision of the discoveries in this field (Frank-Kamenetskaya et al., 2020).

Description of the invention

The present description refers to a particular kit comprising at least one equally particular spongy material employed in the biomedical field and especially for bone regeneration. Specifically, given the complications of the therapies presently used for bone regeneration, the present invention provides that said kit has use, not just in human and animal dental and orthopedic surgery, but also in the field of plastic and maxillofacial surgery. Said spongy material is obtained starting from a hydrogel obtained from secondary derivatives of the food chain. Object of the present description is therefore also a process for obtaining said spongy material or sponge.

In particular the process used and claimed by the Applicant provides for:

- the recovery of pericardia of bovine origin, which are decellularized, lyophilized, reduced into a powder, which is enzymatically treated in order to bring the proteins thereof into aqueous suspension form;

- the aqueous suspension, brought to physiological pH and temperature, becomes a hydrogel, with viscosity greater than that of the starting suspension;

- in order to improve the characteristics thereof, such as the mechanical properties or the compatibility with structures of bone type, other molecules are added during the process of obtaining the hydrogel: hydroxyapatite (HA) and/or hexamethylene diisocyanate (HMDI), the latter acts as cross-linker, in order to reinforce the structure of the hydrogel and improve the mechanical properties thereof. In order to be able to evaluate the effectiveness of these molecules, the hydrogels were attained at different concentrations, both of HA (0%, 60%) and of HMDI (0%, 0.01%, 0.05%, 0.1%);

- in turn, the hydrogels are subsequently lyophilized, so as to obtain dry and spongy structures. the structure of the lyophilized hydrogen thus obtained is not immediately destroyed by the subsequent immersion in a liquid, in order to allow the insertion thereof in the damaged bone structures.

The dimensions of the spongy material or sponge thus obtained are variable but the structure which shapes the fibers of the same is different for each shape. In this manner, the dimensions of the first shape are 1.5-2.0 cm diameter and 2-3 cm height; and those of the second shape are 2.0-3.0 cm diameter and 1.0-1.5 cm height and of the third shape are 3.0- 4.0 cm diameter and 0.5-1 cm height.

Table 1.

The spongy materials of the kit according to the present invention advantageously have the capacity of being imbibed with different autologous fluids of the patient which will increase the biocompatibility and the capacity of regeneration of the tissue, such as platelet-rich plasma, whole blood or hematic fractions. As an alternative, other fluids can be employed such as gels which will functionalize the material for different applications, such as for example the immersion in fibrinogen, conferring hemostatic capacity.

The advantage of being able to modify the porosity and rigidity of the same material by employing crosslinking agents in different amounts offers a versatility in the production chain thereof. Even the lack of crosslinking agent in the material can offer interesting characteristics for several applications, given that it renders the material highly hydrophilic, being completely dissolved in the surrounding aqueous environment.

The protein nature of the structure offers the possibility of adding biomolecules to the material, thus conferring different properties thereto which are linked to the molecules.

Descrrotion of the figures

The invention will be described in more detail hereinbelow with reference to the enclosed figures, in which: FIGURE 1 shows two images of the spongy material which correspond to the first shape A (Fig.l (a)) and to the third shape B (Fig. 1 (b)) Specifically, Figure 1 shows: lyophilized hydrogen with cylindrical form in a first shape A and flat disc in a second shape B.

FIGURE 2 shows a morphological analysis by means of stereoscopic microscope, whose results can be appreciated in Figures 2(a) (first shape A) and 2(b) (third shape B). From said figures, it is possible to observe the modification of the morphology in relation to the quantity of HA and HDMI present, along with the differences between the first shape A and third shape B. Specifically, Figure 2(a) shows the images of the samples at the stereoscopic microscope with various concentrations of HDMI and HA and Figure 2(b) shows the images of the samples at the stereoscopic microscope with various concentrations of HDMI and HA. FIGURE 3 shows the vitality of the cells MG63 cultivated on the sponges for 1, 7 and 14 days. Specifically said figure shows the results obtained in order to evaluate the biocompatibility of the various materials produced in an environment of bone type. In particular, for such purpose, cell lines of osteoblast type were cultivated on the sponges cells, termed MG-63, for times up to 1, 7 and 14 days, and the vitality thereof was evaluated at the end of each of the time points by means of MTS assay (Figure 3(a) and 3(b)).

FIGURE 4 shows the tests of hemocompatibility on the materials. Detailed descriotion of the invention

The invention according to the present description refers to a process for obtaining a spongy material for bone regeneration, which provides for:

- the recovery of pericardia of bovine origin which are decellularized, lyophilized, reduced into a powder, where the powder is enzymatically treated in order to bring the proteins thereof into aqueous suspension form;

- that the aqueous suspension, brought to physiological pH and temperature, becomes a hydrogel, with viscosity greater than that of the starting suspension;

- that in turn, the hydrogels are subsequently lyophilized, so as to obtain dry and spongy structures. Said process can further provide, once the hydrogel is obtained with viscosity greater than the starting viscosity, that during the process of obtaining the hydrogel, other molecules are added that are selected from among: hydroxyapatite (HA) and/or hexamethylene diisocyanate (HMDI), where the latter acts as cross-linker for reinforcing the structure of the hydrogel and improve the mechanical properties thereof. As a non-limiting example HA is added in a quantity of 60% by weight, or HA is added in a quantity of 60% by weight and HMDI in a quantity of 0.01% by weight, or HA is added in a quantity of 60% by weight and HMDI in a quantity of 0.05% by weight, or HA is added in a quantity of 60% by weight and HMDI in a quantity of 0.1% by weight, or only HMDI is added in a quantity of 0.01% by weight, or only HMDI is added in a quantity of 0.05% by weight or only HMDI is added in a quantity of 0.1% by weight.

According to the present description, a spongy material is also described that is obtainable with the aforesaid process, comprising decellularized, lyophilized pericardium of bovine origin, HA, and/or hexamethylene diisocyanate (HMDI) and having diameter comprised between 1.5 cm and 4.0 cm and height comprised between 0.5 cm and 3.0 cm. As a nonlimiting example said material has: diameter comprised between 1.5 cm and 2.0 cm and height comprised between 2 cm and 3 cm; or diameter comprised between 3.0 cm and 4.0 cm and height comprised between 0.5 cm and 1.0 cm. The present description also refers to a kit comprising at least a spongy material as defined above and to the spongy material as defined in any one of the aforesaid examples, as well as to a kit comprising at least one of the aforesaid spongy materials for use as medication for bone regeneration.

In order to be able to have an estimate of the density and of the porosity of the material, a morphology analysis was made by means of stereoscopic microscope, whose results can be appreciated in Figures 2(a) (first shape A; and 2(b) third shape B).

From the figures, one can observed the modification of the morphology in relation to the quantity of HA and HDMI present, as well as the differences between the first and the third shape.

With regard to the wettability of the materials, it was observed that after the immersion in the liquid, the materials maintain the structures thereof obtained after lyophilization and they are not dissolved in the liquid.

In order to evaluate the biocompatibility of the various materials produced in an environment of bone type, cell lines of osteoblast type were cultivated, termed MG-63, for times up to 1, 7 and 14 days, and the vitality thereof was evaluated at the end of each of the time points by means of MTS assay (Figure 3).

From the results, it is possible to infer that the vitality is higher for the cells cultivated on materials which have a low or zero concentration of HDMI, while for the cells cultivated on materials with concentration equal to or greater than 0.05% of HDMI, the vitality decreases. With regard to the effect of the HA concentration on the cellular vitality, in the absence of HDMI, significant differences upon varying the quantity of HA are not encountered.

Both the morphology analyses and those relative to the biocompatibility show that the materials with low concentrations of HDMI and at 0% and 60% of HA possess a cellular vitality suitable for the cellular adhesion and for the cellular repopulation, assisted moxphologically by a presence of porosity suitable for the infiltration of cells and their subsequent nutriment.

From a hematic standpoint, it does not interfere with the normal blood coagulation.

In order to evaluate the behavior of the materials in contact with the blood, coagulation tests were carried out by means of the use of thromboelastography, which calculates different parameters relative to the coagulation starting time, i.e. the reaction time, the coagulation completion time, the speed of formation of the coagulation and the force of formation of the coagulation. The data obtained is compared with the physiological ranges, reported hereinbelow: R: 3.8-9.8 min,

K: 0.7-3.4 min, angle: 47.8-77.7 degrees, MA: 49.7-72.7 mm.

As can be seen from the data reported in Figure 4, the considered parameters fall within the physiological intervals for all the materials, even if encountering several differences for some materials. Indeed, it can be observed that the latter, in which HA and/or HDMI are present, possess values slightly different from the non-enriched sponge.

In summary, it can be confirmed that the modification of the materials does not significantly alter the normal blood coagulation.

Bibliography

Frank-Kamenetskaya, O. V., Vlasov, D. Yu., Panova, E. G., and Lessovaia, S. N. eds. (2020). Processes and Phenomena on the Boundary Between Biogenic and Abiogenic Nature.

Cham: Springer International Publishing doi.lO.1007/978-3-030-21614-6. Hamai, R., Anada, T, and Suzuki, O. (2020). “Novel scaffold composites containing octacalcium phosphate and their role in bone repair , ” in Octacalcium Phosphate

Biomaterials (Elsevier), 121-145. doi:10.1016/B978-0-08-102511-6.00006-6.

Nikolidakis, D., and Jansen, J. A. (2008). Ihe Biology of Platelet-Rich Plasma and Its

Application in Oral Surgery: Literature Review. Tissue Engineering Part B: Reviews 14, 249-258. doi:10.1089/ten.teb.2008.0062.

Rodriguez, I., Sell, S., McCool, J., Saxena, G., Spence, A., and Bowlin, G. (2013). A Preliminary Evaluation of Lyophilized Gelatin Sponges, Enhanced with Platelet-Rich Plasma, Hydroxyapatite and Chitin Whiskers for Bone Regeneration. Cells 2, 244-265. doi.lO.3390/cells2020244.

Claims

1. Process for obtaining a spongy material for bone regeneration, said process providing:

- for the recovery of pericardia of bovine origin which are decellularized, lyophilized, reduced into a powder, said powder being enzymatically treated in order to carry the proteins thereof in aqueous suspension form;

- that in turn, the hydrogels are subsequently lyophilized, so as to obtain dry and spongy structures.

2. Process for obtaining a spongy material for bone regeneration according to the preceding claim wherein it is further provided - once the hydrogel is obtained with viscosity greater than the starting viscosity - that, during the process of obtaining the hydrogel, other molecules are added that are selected from among: hydroxyapatite (HA) and/or hexamethylene diisocyanate (HMDI), the latter acting as a cross-linker in order to reinforce the structure of the hydrogel and improve the mechanical properties thereof.

3. Process according to the preceding claim wherein HA is added in a quantity of 60% by weight.

4. Process according to claim 2 wherein HA is added in a quantity of 60% by weight and HMDI of 0.01% by weight.

5. Process according to claim 2 wherein HA is added in a quantity of 60% by weight and

HMDI in a quantity of 0.05% by weight.

6. Process according to claim 2 wherein HA is added in a quantity of 60% by weight and HMDI in a quantity of 0.1% by weight.

7. Process according to claim 2 wherein HMDI is present in a quantity of 0.01% by weight.

8. Process according to claim 2 wherein HMDI is present in a quantity of 0.05% by weight.

9. Process according to claim 2 wherein HMDI is present in a quantity of 0.1% by weight.

10. Spongy material comprising decellularized, lyophilized pericardium of bovine origin, HA, and/or hexam ethylene diisocyanate (HMDI) and having diameter comprised between 1.5 cm and 4.0 cm and height comprised between 0.5 cm and 3.0 cm.

11. Spongy material according to the preceding claim, having diameter comprised between

1.5 cm and 2.0 cm and height comprised between 2 cm and 3 cm.

12. Spongy material according to claim 10 having diameter comprised between 3.0 cm and 4.0 cm and height comprised between 0.5 cm and 1.0 cm.

13. Kit comprising at least one spongy material as defined in any one of claims 10 to 12.

14. Spongy material according to any one claim 10 to 12 for use as medication for bone regeneration.

15. Kit according to claim 13 for use as medication for bone regeneration.