Hyperelastic bone
Cute Bones 3D: hyperelastic bone material for plastic skull defects
Dmytro_Kikot, Habr
We have about 205 of them, in total they weigh about 5-6 kg and are completely renewed at the cellular level every 10 years. And then there is an idiomatic expression stating that everyone has it in the closet. Of course, we are talking about the skeleton and the bones that make it up. Injuries associated with bone damage are among the most common in the world. Sometimes such injuries do not require treatment of the bone, but its replacement. Bone transplantation is associated with a number of dangers for the patient, including subsequent pain, infection, bleeding, damage to the associated tissues, etc.
Some scientists believe that the key to successful bone transplantation lies in the use of printed bones that will be ideally suited to a particular patient and will be free of defects. How did scientists print bones, what was used for this and what results did the implantation operation on a rat show? We will learn about this from the report of the research group.
The basis of the study
Methods of treating bone injuries have not changed much since the most ancient times. Scientists even analyzed 36 Neanderthal skeletons that showed signs of fractures. Of these, only 11 have treatment of these injuries that can be called unsuccessful.
However, such success of treatment at all times does not apply to all types of fractures. Some injuries managed to be treated successfully and without consequences already in the presence of sufficiently modern medical equipment, knowledge and techniques.
At the moment, bone transplantology often uses autotransplants (fragments of large bones of the patient himself) and allografts (bone fragments obtained from a donor). These methods are quite advanced, but not omnipotent. Cranio-maxillofacial defects (congenital, oncological or traumatic origin) are quite complex. In such cases, patient-specific implants are needed. Therefore, they cannot be taken from a donor, but must be manufactured. In such cases, cranioplasty is used, but the implant will not be able to regenerate and grow together with other bones of the patient. Bone spongy substance, demineralized bone matrix, synthetic bone fragments or bone putties are also used, with which the defect site is filled. But these materials are not porous and have limited bound porosity. Because of this, surface cell migration and vascularization (vascular formation) are reduced, which can lead to encapsulation (capsule formation around a foreign body), rather than tissue integration. As a result, the risk of infection greatly increases. Researchers suggest using three-dimensional printing, because this method allows you to make an inexpensive implant that will be ideally suited for a specific defect in a particular patient.
The researchers also note that biomedical three-dimensional printing still lacks high-performance materials that will combine ease of manufacture and biological functionality. And this means that you need to create your own material.
Hyperelastic bone
And the name of the new material is "hyperelastic bone" (hyperelastic bone material). This osteoregenerative material is produced by extrusion at room temperature of a mixture of hydroxyapatite (solid, 90% of the total mass) and poly (lactic glycolic) acid (liquid, 10% of the total mass) into three-dimensional shapes without the need for sintering, curing or other forms of physico-chemical stabilization.
The resulting three-dimensional printed frame has good elasticity and high absorption. In addition, the skeleton causes osteogenic differentiation of human mesenchymal stem cells obtained from the bone marrow without the addition of osteoinducing catalysts. At the same time, the hyperelastic bone did not cause a negative immune response, became vascularized and integrated with the surrounding tissues, supporting the growth of a new bone. Another achievement is the possibility of transferring transduced human fat stem cells by means of a printed implant.
Next, we will get acquainted with the results of the practical test in more detail. Scientists conducted a comparative analysis of the osteoregenerative ability of hyperelastic bone and a commercial variant (autologous bone) with skull defects of critical size in rats. But first, a little bit about the preparation for the experiment and how exactly the test material and the implant were made.
Manufacture of hyperelastic bone
As we already know, the hyperelastic bone frame was made of hydroxyapatite and poly (lactic glycolic) acid.
BioPlotter Printer
All samples were printed using a BioPlotter Manufacturing printer from EnvisionTEC. The thickness of the sheets (5x5 cm), consisting of 5 layers of 120 microns, amounted to a total of 0.6 mm. Then round (8 mm in diameter) blanks were squeezed out of the sheets using a stylet for biopsy. The resulting blanks were washed and sterilized.
Image No. 1: the process of manufacturing implants.
Implantation surgery
Laboratory Sprag-Dole male rats weighing approximately 500 grams each acted as test subjects.
During the operation, the subjects were under general anesthesia (2% isoflurane / 100% oxygen). A sagittal incision (1.5 cm) was made between the lambdoid and coronal sutures to expose the skull. With the help of a hand drill with a trepan (a needle with a milling cutter or a drill for forming holes in dense tissues), an artificial skull defect of 8 mm in diameter was created.
The subjects were divided into 4 groups:
7 individuals – negative control group (without an implant on the defect);
6 individuals – positive control group (with an autologous bone as an implant);
6 individuals – study group No. 1 (with a poly (lactic-glycolic) acid frame as an implant);
10 individuals – study group No. 2 (with hyperelastic bone as an implant).
Image No. 2 shows a photo of the test subjects during the operation (please do not look at the faint of heart, the process was described in text form earlier).
After implantation (or without it), the periosteum and skin were closed using a movable absorbable suture, and an anesthetic was administered to the subjects. The subjects were kept in cages by two. Access to water and food was unlimited.
Skull samples were analyzed by cone-beam computed tomography. The areas of interest were cut out of the skull bone, placed in 70% ethanol and scanned using a micro-CT scanner. Then, using the software for analyzing medical images (Mimics Medical 19.0), the scientists examined in more detail the areas of the skull where the implants were implanted.
After micro-computed tomography, the samples were cut in half for histological analysis and visualized using scanning electron microscopy. The degree of regeneration was also assessed after 8 and 12 weeks.
And now we will proceed directly to the results of observations.
Research results
Image No. 3a: gray – with autologous bone; black – without an implant; blue – with an implant made of poly (lactic glycolic) acid; red - with hyperelastic bone.
The image above (3a) shows three-dimensional reconstructions of cone-beam and micro-computed tomography. The amount of regenerated bone tissue was determined by the amount of mineralized bone as a fraction of the total volume of tissue of interest. The bone volume per share of the total volume for hyperelastic bone, poly (lactic glycolic) acid and for the negative control group were normalized relative to the bone volume per share of the total volume for the positive control group (with autologous bone). Thus, a comparative analysis of the performance indicators of all implant variants was carried out.
Image #3b
Cone beam and micro-computed tomography showed an increase in the amount of mineralized bone matrix in defects treated with hyperelastic bone implants (3b).
According to cone-beam tomography, the volume of mineralized bone in the case of using a hyperelastic bone implant was 55.7% at week 8 and 57.0% at week 12 of observations. According to micro-computed tomography – 36.1% at week 8 and 37.1% at week 12 of observations. This is the data before normalization.
After that, normalization was carried out to the indicators of the volume of mineralized bone in the case of autologous transplants. Now it became clear that the volume of regeneration using hyperelastic bone was 95.6% and 82.0% (8 and 12 weeks of observations) of the volume of the positive control group (with autologous bone). And micro-computed tomography gave the following results: 74.2% and 64.5% (8 and 12 weeks of observations).
The use of exclusively poly (lactic-glycolic) acid as a material for implantation turned out to be quite ineffective: 16.6% and 22.5% (8 and 12 weeks of observations) of the volume of the positive control group. The ineffectiveness of this method is also confirmed by the fact that its results do not differ much from the results of the negative control group, which had no implants at all: 10.3% and 13.8% on cone-beam tomography and 14.5% and 19.5% on micro-computed tomography.
Comparison of the results of the tested new material (hyperelastic bone) with the results of the negative control group showed a difference in the volume of mineralized bone by 7.81 times at week 8 and 5.75 times at week 12 in favor of hyperelastic bone.
Thus, in terms of the volume of regeneration, the use of hyperelastic bone is practically comparable to the use of commercial variants of implants with autologous bone.
Image No. 4: arrows – defective edge; Ft – fibrous (fibrous) tissues; Mc – membrane-cell component; Nb – new bone.
The histological analysis only confirmed the data of cone-beam and micro-computed tomography. The scientists identified the edges of the defects, and the formations of the new bone were specially stained with eosin for better visualization.
In the case of the negative control group, fibrous tissue was observed, but the formation of new bone was minimal (the upper row in image No. 4). Samples with poly (lactic glycolic) acid also could not boast of a large volume of newly formed bone (third row).
But the samples where hyperelastic bone was used, on the contrary, showed the formation of mineralized bone tissue on the surface of the edges of defects (row 4). At the 8th week of observations, fibrous tissue and membrane-cellular components inside the implant appear in the places of defects, and at the 12th week, the formation of a new bone begins around the elements of the implant frame.
Image #5
And finally, the analysis of SEM (scanning electron microscope) images of samples with hyperelastic bone at the 12th week of observations showed the formation of close cellular contact of tissues with the material inside the implant.
For a more detailed acquaintance with the nuances of the study, I recommend taking a look at the report of scientists published in the journal Plastic and Reconstructive Surgery (Huang et al., Three-Dimensionally Printed Hyperelastic Bone Scaffolds Accelerate Bone Regeneration in Critical-Size Calvarian Bone Defects).
Epilogue
In this work, scientists have demonstrated a new type of osteogenic biomaterials that allow the creation of implants for the treatment of bone defects. Scientists call the most important features of their brainchild: simplicity of implantation, ease of manufacture, high efficiency, low cost of production and the possibility of personal adjustment of the implant to a specific patient.
Hyperelastic bone is really very elastic and can take the necessary shape both during manufacture and at the time of implantation, which greatly facilitates this process. Ceramic and polymer-ceramic implants cannot boast of this.
But even this is not the most important advantage. A high degree of bone regeneration and implant survival is much more important. Already 4 weeks after implantation, the active process of bone mineralization begins.
Scientists also note that such speed and efficiency are extremely important in the case of sufficiently large defects (as it was demonstrated during practical experiments).
The use of such technology can greatly simplify the lives of both doctors and patients. The individuality of treatment, the speed of production, implantation and rapid recovery without side effects is an excellent advertisement for the new technology. In the future, scientists intend to conduct several more experiments, study the regeneration process in more detail and improve their invention.
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