Biological performance of a bioabsorbable Poly (L-Lactic Acid) produced in polymerization unit: in vivo studies

Study objective

This work aims to evaluate the in vivo biocompatibility of biomaterials synthesized from the polymerization of Lactide monomers and compare the final synthesis cost to the PLLA biopolymer import price.

Manufacturing of PLLA

Poly-lactic acid synthesis. The synthesis of PLLA was conducted by bulk polymerization by adding L-lactide monomer into a glass reactor containing the catalyst Sn (Oct) 2 (Sigma). The proportion monomer/catalyst was 0.5%. The mixture was immersed in an oil bath at 140°C for 2 hours under nitrogen flow. The produced polymer was dissolved in chloroform, CHCl3 (Merck), precipitated in ethyl alcohol and dried in a vacuum oven at 60°C for 12 hours. After the synthesis were manufacture tablets of PLLA.

Manufacturing PLLA tablet. The scaffold size was determined by reviewing previously published studies that determine the size of critical lesions for osseointegration and biocompatibility studies in rabbits^18,19. PLLA tablets (Figure 2) of 3mm × 15mm were produced by compression of the polymerized raw material under a hydraulic press.

Figure 2.

A – 3D representative image and dimensions of the PLLA tablet. B - photo of a compacted PLLA pellet, ready for insertion into a rabbit lesion.

In vivo experiments

Study design. The animals were selected and divided into groups, at the post-surgical time determined for each group the initial number of animals was not changed as shown in the study flow diagram.

Experimental models - eligibility criteria. 12 New Zealand female rabbits (age = 16–20 weeks) were assigned to two groups:(1) Injury + PLLA group (n = 6), (2) Injury no PLLA group (n = 6). Each group was divided into two subgroups at six and nine months post-surgical procedure. Rabbits were kept in individual cages with food (PROVIFE, Argentina) and water ad libitum. All procedures were made according to the approval of the ethics committee nº44371/0028 of 1MED423- University National of Rosary, Argentina. Their regulations are in accordance with well-established guidelines for the care and handling of animals to minimize animal pain and suffering according to the 3Rs (replacement, reduction and refinement) and follow international regulations for the care and use of laboratory animals. All efforts were made to ameliorate harm to animals

Pre-surgical preparation. Antibiotic prophylaxis and anesthetic treatment were performed. Before the surgical procedure, rabbits received antibiotic prophylaxis Cefazolin 50mg/Kg/day (administered intramuscularly). Anesthetic treatment was performed using a combination of two drugs administered intramuscularly: ketamine hydrochloride 35mg/kg animal, xylazine hydrochloride 2% 18mg/kg animal, and acepromazine maleate 1mg/kg animal. The anesthetic effect lasted 45 – 60 minutes.

Surgical procedure. The surgeries were performed under aseptic conditions. A longitudinal incision of 3 cm total length was made parallel to the sagittal suture below the Lambdoid suture (5–6 cm long). The central perfuration point is marked with a surgical marker, the drilling was done with a 15 mm drill, made of stainless steel with laser engraving, generating a hole of 15 mm in diameter and +/-3 mm in depth. After dissection of the periosteum, the defect was made in the center of the sagittal suture, 2 mm below the Lambda suture. In one group of animals PLLA was introduced in the center of the lesion (Lesion + PLLA Group) and in the other group the bone lesion was maintained (Lesion without PLLA Group) (Figure 4).

Post-surgical procedure. The rabbits were clinically monitored daily in basis as to overall status, mobility and food intake. Post-surgical analgesia was performed using tramadol every 12 hours, (6 mg / kg / day) intramuscularly during three days. Antibiotic cephalexin, 50mg/kg/day and anti-inflammatory meloxicam 0,75mg/kg/day intramuscularly on the day of surgery and 3 days following surgery.

Biochemistry studies

Hemogram. Blood samples were collected before sacrifice. The blood samples were collected in EDTA tubes to perform hemogram, after homogenization of the samples. Red blood cells (RBC), white blood cells (WBC), hemoglobin (Hb), hematocrit (Hto), mean corpuscular volume (MCV) and platelets were evaluated.

Transaminases. The serological study was performed on blood samples without anticoagulant, centrifuged for 10 minutes at 10,000 rpm, to separate the serum fraction from the blood, where the values of the enzymes transaminases, glutamate-oxalacetic transaminase (GOT) and glutamate pyruvate transaminase (GPT) were determined. All determinations were performed with Wiener lab Kids (Argentina).

Biochemical statistical evaluation was performed with non-parametric analysis of variance (Wilcoxon). A p value <0.05 was considered significant

Animal euthanasia and sample collection

The animals were euthanized in the 50 % of each group at six-month post-surgery and the others at nine months, with an intraperitoneal injection of five times the value of the anesthesia dose. Then, cranial were obtained by axially cutting the neck near the base of the skull with a bench saw, which were immediately used for tomographic, macroscopy and histology studies.

Macroscopy observation

Prior to sacrifice, each implanted area was inspected. Surface characteristics such as skin color, scarring, edema, erythema, and elevations were evaluated. A sagittal incision was then made at the scar site and the lesion area was cut with a 5 mm margin from the edges to remove a portion of bone with the central lesion. After removal of the skull fragment containing the central lesion, the endocranial and exocranial side of the fragment was evaluated for edema, surface characteristic, implant position, exudate or other signs of infection.

Tomographic studies

Tomographic studies were performed on the day of sacrifice of the group of animals, each skull obtained from the axial section was immediately used for tomographic study with a 16-channel Toshiba multislice CT scanner for qualitative analysis of the characteristics of the lesion edge and the progression of the tissue around the biomaterial.

Histological analysis

After euthanasia, tissue preparation was conducted according to a previously described method. Briefly, the lesion area was explanted with a 5 mm margin around, fixed in 4% formaldehyde for 24 hours and then dehydrated in a series of increasing concentrations of alcohol and xylene. After that, the samples were serially cut (5 µm thick) using a manual rotary microtome (Micron-Zeiss, Germany), and stained with hematoxylin and eosin and by Masson trichrome staining. All samples were examined by light microscopy and evaluated. Photomicrographs were taken from slides of each specimen using a digital camera mounted on an Olympus CH30 microscope.

Osteoregenerative analysis

Studies were conducted to consider if the proportion of regenerated bone in animals in the group implanted with the scaffold under study was greater relative to animals in the group not implanted with PLLA at each of the time points considered for this experiment, six months and nine months, respectively.

Three histological sections were studied for each animal in each of which three sites were randomly selected. ImageJ software was used for the observation of de novo regenerated bone. The New bone formation was quantified and expressed as a percentage of total bone (% BV/TV) in the area surrounding the lesion.

Data were analyzed using linear mixed models implemented in Infostat software. Outliers were removed before the analysis was performed.

Statistical analyses: The variable of interest is worked out in original scale, in proportion between 0 and 1. To detect significant differences between Groups, linear mixed models²⁰ were fitted and the Bayesian information criterion²¹ was used to select the best fit. Fisher's a posteriori test was used with a significance level of 0.05. The software used for the analysis was Infostat²². These procedures were followed for both the evaluation at six months and nine months and between six months.

Production and import cost analysis

The cost of the raw material needed to synthesize the biomaterial from the lactide monomer was compared with the cost of importing the PLLA polymer. The currency conversion of the prices in dollars (US$) to real (R$) was performed. To analyze the cost of synthesizing 1 gram of PLLA, the price of 100g of lactide monomer, dudecanol, octanoate and the Nitrogen (N2) cylinder was obtained, and then the cost of each quantity used in the synthesis was determined. The costs of each material were added up to obtain the price of 1g of synthetized polymer. This value was compared to the cost of 1g of the imported material by dividing the price of the material by the content of 100 grams of the package. (Figure 3)

Figure 3. Blue square representing cost calculations of PLLA synthesis.

Violet square representing cost calculations of each gram of already synthesized imported PLLA. Yellow square represents comparison of costs of laboratory synthesized material and imported biomaterial.

Manufacturing of PLLA- Poly-lactic acid synthesis

The PLLA was synthesized by opening of the cyclic dimer of L-lactide in order to obtain high molecular weight polymer. The synthesis temperature was maintained at 140 °C/ thus avoiding, high temperatures, which lead to a depolymerization process which allows the decrease of the molecular weight of the polymer²³.

The obtained polymer had the PLLA molecular weight 86.93 g/mol as mentioned in previous works¹⁰.

Surgical Procedure

As can be seen in Figure 4, the surgical procedure with the selected drill allowed the achievement of a central lesion with a diameter considered as a critical lesion, and it was also possible for the biopolymer to be placed in the center in order to analyze its biocompatibility with the edge of the bone in question in the group (Injuria +PLLA).

Figure 4. Surgical Procedure.

A 3-cm full-thickness longitudinal incision (A). The central drilling point (B). Defect in the center of the sagittal suture, 2mm below the Lambda suture (C).

In Figure 4-A, after asepsis with iodopovidone and covering the surgical area on top of the head, a sagittal cut was made with a scalpel. After removal of the periosteum, the start of the circumferential lesion can be seen through a 15mm trephine, before the central bone is removed (Figure 4-B). After total removal of the central bone, a part of the brain can be seen in Figure 4-C, covered by the meninges.

Biochemestry results

Hemogram. The hemogram statistical studies showed that there were no significant differences between the groups at any of the times studied, in any of the variables considered. In Figure 5 and Figure 6 we can observe the results obtained the day before euthanasia. Each of the results obtained for each variable applied to each sample, were always within normal ranges for animals of this line and age²⁴

Figure 5. In this figure we have the graphs representing the values of the different variables measured in the blood count study of the animals under study, in the period of 6 months post-surgery.

1: Lesion + PLLA group. 2: Lesion No PLLA group.

Figure 6. In this figure we have the graphs representing the values of the different variables measured in the blood count study of the animals under study, in the period of 9 months post-surgery.

1: Lesion + PLLA group. 2: Lesion No PLLA group.

Transaminases. Statistical studies of transaminase levels showed that there were no significant differences between the groups for any of the variables considered. Each of the results obtained for each variable applied to each sample (Figure 7).

Figure 7. In these graphs are represented the values for the different variables measured in the transaminasas study of the animals under study, in both period post-surgery 6 and 9 months.

Group 1: Injury No PLLA. Group 2: Injury + PLLA.

Tomographic results

The images observed on the CT scans allowed an analysis of the lesion border in the Injury+PLLA group and the Injury No PLLA group.

In the Injury No PLLA group at 6 and 9 months, it is possible to observe the formation of a small fragmented layer of bone tissue over the meninges, however, there are no differences in bone formation activity at the edge of the lesion. The analyses of the animals that have the PLLA implant in their lesion, showed hyperdense borders with signs of bone regeneration. In addition, it can be seen that under and above the implant there are hyperdense sectors, also indicating the formation of a layer of bone tissue around the implanted matrix. (Figure 8)

Figure 8. Representative tomographic images of animals at 6 and 9 months post implantation.

The larger images represent a coronal slice and the smaller images in the lower right corner represent axial cuts. On the left images, represent the animals that did not receive the PLLA implant, where the red line shows the lesion area. The right images represent animals that received PLLA implantation in the center of the lesion (yellow arrow) and the red line shows the lesion area.

Macroscopy observation results

After sectioning the area with the central lesion, a clinical presentation of the groups examined at six and nine months is shown in Figure 9. A fibrous scar covered the entire area of the defect. From the Exocraneal view much of the polymer can be visualized below a thin layer of subcutaneous tissue. In the endocranial view it was covered by the adhered meninges, where the biopolymer could hardly be assessed. No apparent indications of infection were detected in any of the experimental groups macroscopically.

Figure 9.

Cranial gross appearance of the defects, showing the fibrous scar covering the defect area (A), exocranial (B) and endocranial face (C).

Histological results

Hematoxilin and eosin. In the hematoxylin and eosin staining results, at a 10X magnification it is possible to analyze the cell types present at the margin of the lesion and compare them with the group of animals that did not receive the PLLA biopolymer implant. In the six-month-old animals without implantation, it is possible to observe the scarce formation of connective tissue and some inflammatory cells and the irregular and undefined border of the lesion.

When analyzing the six-month-old animals with bone implant, there is a thick layer of connective tissue at the margin of the biomaterial, with an evident and well-defined border. In the region of contact with the polymer, groups of inflammatory cells can be seen around the fragments of biomaterial. In the surrounding connective tissue, besides fibroblasts, it is possible to see bone tissue formation, with the presence of osteoblasts, osteocytes and osteoclasts. (Figure 10 – A and C)

Figure 10.

HE staining- original magnification 10x – A 6 months, Injury No PLLA animal. B nine months, Injury No PLLA animal. C six months, Injury + PLLA animal. D 9 months, Injury + PLLA animal. Bone is presented as a compact structure in a pink color, connective tissue in light pink and the scaffold structure (PLLA) in light grey. OSB, osteoblast; OTC, osteoclast; OST, osteocyte; FB, fibroblast; BV, blood vessel; NB, new bone and Defect Margin indicates edge the tissue boundary.

When comparing and analyzing the nine-month-old animals, it is possible to verify that the animals with a critical lesion without implant have an increased layer of connective tissue around the edge of the lesion, the presence of adipose tissue, and few inflammatory cells. In the animals nine months old with implants, the plaque fragments are surrounded by phagocytic cells, surrounded by abundant connective tissue and new bone tissue, and abundant osteoblastic and osteoclastic cells can be seen. Blood vessels are also present and distributed around the PLLA biopolymer. In none of the animals is there the formation of thick connective tissue suggestive of fibrous capsule. (Figure 10 – B and D)

When analyzed at 40x magnification, the cell types present at the edge of the lesion are observed. The six-month-old animals with lesions without polymer have the formation of abundant dense connective tissue, without signs of intense inflammation or signs that indicate regenerating cellular activity such as the presence of new bone tissue. When compared to the six-month-old animals, at the same 40x magnification, we see the presence of inflammatory tissue surrounded by fragments of polymer and also abundant blood vessels throughout the area.

Masson's trichrome

The collagen is seen in blue or greenish color, while the bone acquires a red color. through this differentiation we can delimit the color margin in the ImageJ program, quantifying the percentage of bone present at the edge of the lesion and comparing the different groups and times.

After Masson staining and quantification of the area in pixels represented by dark blue and red colors, the area of bone growth bordering the lesion can be seen. In a qualitative analysis of the Masson staining, it can be inferred, together with what was observed in the hematoxylin and eosin staining, that the animals implanted with PLLA present a greater amount of bone tissue surrounding the lesion.

Together with the images, we can analyze below the statistical data that represents the area of bone and connective tissue of the analyzed groups (Figure 12)

Figure 12. Masson Staining - original magnification 10x.

-Histomorphometric analysis of connective tissue lesion area and bone tissue area in six- and nine-month-old animals with and without PLLA. First row with original photos at 10X magnification with Masson staining. Second row with total tissue area in red by image analysis software image J. Third row representing bone tissue area in red.

1. Osteoregenerative statistical results

Significant differences were detected between the groups at both six and nine months of post-surgical evaluation. The Injury+PLLA group achieved 62% bone regeneration at six months, while the Lesion+PLLA group achieved only 30% bone regeneration (Graph 2). At nine months of evaluation, the Injury+PLLA Group achieved 66% of both regenerations, compared to 31% in the Injury No. PLLA Group (Graph 3).

Graph 2. Difference of adjusted means for treatments: PLLA 6 month and Control 6 month.

Different letters indicate significant differences in LSD Fisher test with a significance of 0.05.

Graph 3. Difference of adjusted means for treatments: PLLA 9 month and Control 9 month.

Different letters indicate significant differences in LSD Fisher test with a significance of 0.05.

Production and import cost analysis results

The prices of the raw material for the polymerization of the biomaterial in the laboratory were obtained through quotations in Sigma Aldrich. and Corbion.

The price of synthesizing 50g of PLLA was first calculated by dividing the value of R$7. 559.00 per 50 grams of the lactide monomer (1kg) Purasorb L, R$1413.00 of the Tin Octanoate (1kg) divided by the 0.75 grams needed, R$138.00 of the CHCl3 (1L) cost was divided by the 0.00057ml used and the nitrogen used for 150 minutes of reaction corresponds to 3 liters, where the cost per liter of N2 was calculated by dividing the R$143.60 by the 10L and then multiplied by the 3 liters needed. Once the cost of producing 50 grams of polymer was obtained, this cost was divided by the value of 50 and the cost for each gram of polymer synthesized in the laboratory was found (Table 1).

The price of the imported PLLA polymer was quoted by two companies: TCI America and ACROS Organics. The packages contained 25g of PLLA polymer each. The cost per gram of imported polymer was calculated by dividing the package price by 25, where the costs found for each brand were: R$11.49/g of PLLA polymer (L-Lactide 98.0+%, TCI America™) and R$13.92 of the polymer (L-Lactide, 98%, ACROS Organics™) (Table 2).

Comparing the costs for each imported gram of PLLA of R$11.49 and R$13.92 to the obtained value of R$8.43 for laboratory synthesized PLLA, a difference of R$3.06 is calculated between synthesized PLLA and imported PLLA (TCI America™) and a difference of R$5.49 for laboratory synthesized PLLA compared to imported PLLA (ACROS Organic™).

Underlying data

Zenodo: ARRIVE Questionnaire, CONSORT Check-list and Flow Diagram + TGO, TGP, HEMOGRAMA AND OSTEOREGENERATIVE DATA - Paper: "Biological performance of a bioabsorbable Poly (L-Lactic Acid) produced in polymerization unit: in vivo studies", https://doi.org/10.5281/zenodo.5396921²³.

This project contains the following underlying data:

Zenodo: Images, graphs and tables from the article: Biological performance of a bioabsorbable Poly (L-Lactic Acid) produced in polymerization unit: in vivo studies - https://doi.org/10.5281/zenodo.5216707.

This project contains the following underlying data:

Zenodo: Biological performance of a bioabsorbable Poly (L-Lactic Acid) produced in polymerization unit: in vivo studies - HEMATOXYLIN&EOSIN, MASSON AND CT SCAN IMAGES, https://doi.org/10.5281/zenodo.5396584.

Reporting guidelines

Zenodo: ARRIVE Questionnaire, CONSORT Check-list and Flow Diagram + TGO, TGP, HEMOGRAMA AND OSTEOREGENERATIVE DATA - Paper: "Biological performance of a bioabsorbable Poly (L-Lactic Acid) produced in polymerization unit: in vivo studies", https://doi.org/10.5281/zenodo.5396921.

This project contains the following reporting guidelines:

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).