BIOCOMPATIBILITY SAFETY ASSESSMENT

Tests performed at the laboratory of preclinical and surgical studies at the IRCCS Rizzoli Orthopaedics Institute, Bologna, Italy

All safety and efficacy tests in vitro and in vivo have been carried out in compliance with current regulations for Medical Devices (ISO 10993-1:2010):

Dermal irritation: tests performed on three albino rabbits applying the product in two areas on the back of each animal and observed at 24, 48 and 72 hour intervals. No signs of erythema or oedema in any animals; According to the results obtained the Primary Irritation Index (PII) resulted as 0.278 and the irritation classification is “non-irritant” (Table 1 PII result classification for rabbit skin irritation)

Table 1 PII response classification for irritation in rabbits. The Primary Irritation Index obtained (equal to 0,278) is classified as “non-irritant”.

Delayed hypersensitivity: test performed on 15 guinea pigs (10 for experimental material and 5 for control group) with intradermal injections of the materials in 2 sites for each solution; the material under testing did not cause any allergic reaction of hypersensitivity when observed after 24 and 48 hour intervals (table 2).

Table 2 Points assigned to each animal in the test group after 24 and 48-hour observations according to the Magnusson and Kligman scale. (where 0 = No visible reaction).

Acute intravenous systemic toxicity: test performed on 15 mice observed at 4, 24, 48 and 72-hour intervals after injecting material extract. Absence of negative physical symptoms and weight change.

Subacute systemic toxicity: this test was performed on two species of animal (rat and rabbit) using two different injection methods (subcutaneous and intramedullary injection).
1st test performed on 10 rats using subcutaneous hydrogel injections. Observations after 4 weeks showed no signs of weight change or clinical symptoms. Despite these results, biochemical and coagulative blood tests were performed as well as histological analysis but no pathological alterations were discovered.
2nd test performed on six rabbits by injecting the experimental material into the intramedullary canal of the femur. Observations after 4 weeks showed no signs of weight change, clinical symptoms, or negative blood biochemical alterations.

Sub chronic systemic toxicity: test performed on 20 rats using subcutaneous hydrogel injections. Observations after 12 weeks. Absence of weight change and clinical symptoms.
Despite these results, biochemical and coagulative blood tests were performed as well as histological analysis but no pathological alterations were discovered.

IN VIVO BIOCOMPATIBILITY

Research carried out at the Department of Orthopaedics, University Medical Center Utrecht, Utrecht,
The Netherlands (1)

All safety and efficacy tests in vitro and in vivo have been carried out in compliance with current regulations for Medical Devices (ISO 10993-1:2010):

Local reactions to in vivo implant
Research was carried out on 10 rabbits to assess the local reactions to hydrogel injected into the intramedullary canal of the femur. Experimental time frame: 12 weeks. DAC® hydrogel was injected into the right femur, while the control hydrogel (hyaluronic acid-based implantable device) was injected into the left femur.

Results: DAC® did not cause bone inflammation reactions and/or any degenerative processes in bone tissue at intramedullary canal level. Histomorphometric analysis did not reveal any significant differences between DAC® and the control hydrogel.

Conclusions: no structural or histological alteration at cortical and trabecular bone tissue level in the intramedullary cavity (Figure 1 and Table 3).

Figure 1 (a, b, c, d)
Histological images of certain frontal sections of the right lateral condyle. Enlarged. 0.3x – Toluidine blue, Fuchsine acid and Fast-green coloration
Table 3
Histomorphometric analysis did not reveal any statistically significant difference between DAC® and the control material with a widely accepted clinical and biocompatibility profile

Effect on implant osseointegration
Histological profile of rabbit tibiae (coloration using basic Fuchsine and Methylene blue) implanted with an intramedullary nail (black circle), coated with DAC®.
Four weeks after the implant it was observed that the DAC® coating had caused no interference with the local osseointegration.

Conclusions: DAC® does not provoke any structural or histological alterations at cortical and trabecular bone tissue level in the endomedullary cavity. No interference was observed with the osseointegration in the animal model.

In vitro biocompatibility
(Research carried out at the IRCCS Rizzoli Orthopaedics Institute, Bologna, Italy)
Cytotoxicity – Genotoxicity: the material resulted as being non-cytotoxic, non-genotoxic and non-mutagenic.

In vitro degradation
(Research carried out at the Novagenit Research laboratories, Mezzolombardo – TN – Italy)
The material was reabsorbed a few days after the implant. The molecules resulting from degradation were hyaluronic acid and poly-lactic acid that are well-known bio-absorbable materials.

EFFICACY

Efficacy with in vivo peri-implant infection
Research carried out at the laboratory of preclinical and surgical studies at the IRCCS Rizzoli Orthopaedics Institute, Bologna, Italy

a)    Local anti-infective effect of hydrogel combined with antibiotics after contamination with 0.2 x 106 cfu

  • Animal model (rabbit)
  • Systemic prophylaxis with Vancomycin
  • Wild-type bacterial strain inoculation of Methicillin-resistant Staphylococcus aureus (MRSA) 106 cfu
  • Groups
    Sandblasted titanium nail + DAC®
    Sandblasted titanium nail + DAC® + 2% Vancomycin
    Sandblasted titanium nail + DAC® + 5% Vancomycin
  • Time frame: 7 days

Results: after 7 days the hydrogel combined with Vancomycin reduced the bacterial load by up to 99.9% compared to the control group. Hydrogel combined with Vancomycin prevents the systemic diffusion of local infection (table 4)

Conclusions: the hydrogel inhibits local infection in vivo. (2)

Table 4
Reduction of the final bacterial load in groups 2 and 3 seven days after contamination with 0.2 x 106. Swab: intramedullary canal  swab – Bone: bone fragment  –  Nail: intramedullary nail

b)    Systemic effect of hydrogel combined with antibiotics after contamination with high bacterial load 

As well as the local effect, it was found that the hydrogel combined with Vancomycin is efficacious in inhibiting the development of a systemic infection with loads of 0.2 x 106 cfu.
The application of the hydrogel combined with Vancomycin even at a concentration of less than 2% (w/v), is highly efficacious in blocking the development of a systemic infection even with very high initial bacterial loads.

Table 5  Systemic bacterial load 7 days after contamination with bacterial loads of 0.2×106 and 0.2×104 cfu.
Emo+ blood culture under aerobic conditions
Emo– blood culture under anaerobic conditions

c)     Local anti-infective effect of hydrogel combined with antibiotics after contamination with 105 cfu

The graph shows the effect of the DAC® hydrogel coating combined with 5% Vancomycin, in the prevention of infections induced by a strong intraoperative inoculum (105cfu that represents a worst case experimental condition) of Staphylococcus aureus (Wood 46—ATCC 10832) (1).

This research carried out at the University of Utrecht, was aimed at demonstrating the comparison between two groups of rabbits.

  • 1st group: no hydrogel DAC®
  • 2nd group: hydrogel DAC® + 5% Vancomycin

To represent an even further “worst case” experimental condition, no groups were administered systemic prophylactic antibiotics (which is normally established practice).
All rabbits received a sandblasted titanium intramedullary nail implant in the tibia. The tibia cavity was contaminated with a strong inoculum of 100 microlitres of solution containing 105 cfu of S. aureus immediately prior to the implant.
After 28 days it was observed that the “DAC® hydrogel + Vancomycin group showed no alterations in the serosanguineous parameters (neutrophil count, erythrocyte sedimentation rate (ESR) or weight loss.

Except for one example, the rabbits in the “no DAC® hydrogel “ group showed positive signs of bacteria in the tibia area, while none of the rabbits in the “DAC® hydrogel + Vancomycin” group had positive bacterial culture.
The difference between the hydrogel + Vancomycin group and the nail implant group without coating was statistically significant (P = 0.01, two tailed Fisher’s exact test).

Synergistic antibiotic effect. In vitro testing.

The minimum inhibitory concentration (MIC) of the compounds tested in combination with DAC® resulted as being reduced demonstrating the synergistic effect of the association between the hydrogel and the compounds examined (table 6).

Table 6 MIC of certain compounds tested in combination with DAC®

Used in combination with different antibacterial drugs DAC® demonstrated greater antibacterial action than the respective antibiotics when employed alone (1).

The antibacterial and antibiofilm action of DAC® (studied using the Christensen method) (3) combined with different drugs, verified during preliminary testing and recorded in table 7 (1).  Data figures coincide for both Staphylococcus aureus, for Staphylococcus epidermidis, and on the sandblasted titanium surfaces, Co-Cr or PE (table 7). 

Table 7 Staphylococcus aureus growth inhibited by DAC® gel combined with various drugs
(experiments on titanium disks with an average of three repetitions)

Vancomycin release in vitro
Tests performed at:     a) Novagenit Research Laboratories, Mezzolombardo – TN – Italy
b) Laboratory of Clinical Chemistry and Microbiology at the IRCCS Galeazzi Orthopaedic Institute Milan, and the University Hospital of Larissa, Larissa, Greece

Release kinetics of different substances were assessed in vitro using both analytical quantification and microbiological tests.

a) DAC® loaded with 2% Vancomycin released approximately 60% of the antibiotic during the first 4 hours, and approx. 80% after 24 hours.

b) The release kinetics coincided with a wide range of antibacterial molecules and several types of substrate (titanium, cobalt chromium alloy, polyethylene).
Complete release in vitro occurs within 48 hours (Figure A, B, D, E)

Release research in vitro

Spectrophotometric and microbiological methods were used on different surfaces (cobalt chromium, Co-Cr; polyethylene, PE; sandblasted titanium, Ti)

Antibiofilm action by DAC® hydrogel combined with Vancomycin or Gentamicin on strains of Staphylococcus aureus or Staphylococcus epidermidis (horizontal coordinate time measured in hours). The combined association presents greater antibiofilm action compared to Vancomycin or Gentamicin employed alone (P<0.05) in every tested time span interval. In all cases, the release peak was observed 2-4 hours after application, regardless of the compound under assessment or the various materials being tested. Release of most of the compounds was complete after 48 hours.

Adherence in vitro and in vivo
Tests performed at:     a) Reconstructive Surgery and Bone and Joint Infection Centre at the IRCCS Galeazzi Orthopaedic Institute, Milan, and the Department of Orthopaedics, University Medical Centre Utrecht, Utrecht, The Netherlands (1)
b) Department of Orthopaedics, University Medical Centre, Utrecht, The Netherlands

In order to assess the permanence and adhesion of the DAC® coating including following insertion using the “press-fit” technique for a bone implant, tests were performed on human cadaver femurs (a) and on animal models (b) using the same experimental procedure.

Figures a1-5. Scrape test: After the scrape test performed with DAC® hydrogel coloured with Methylene blue after the implant of a “press-fit” sandblasted titanium prosthesis, it was observed that more than 80% of the hydrogel was found on the implant following the 180-degree opening of the human cadaver femur.

Figures b1-2: The test on the animal model (test performed on six rabbits) with a sandblasted titanium intramedullary nail implant in the tibia, it was observed that:

•    Approximately 70% of the DAC® coating was attached to the implant while the remaining gel was on the internal surface of the tibia in direct contact with the implant.

•    The distribution of the gel appeared homogeneous along the total length of the implant.

INSTRUCTIONS AND CLINICAL APPLICATIONS

When appropriately combined with antibiotic drugs, DAC® is able to prevent or considerably reduce the formation of bacterial biofilm and bacterial colonisation on implantable materials, reducing the danger of post-surgical infection in animal models (1, 2)

DAC® hydrogel combined with an antibiotic is indicated for patients undergoing surgery for joint replacement or receiving fixation devices for fractures, where additional protection is necessary against possible associated infection.

DAC® hydrogel is particularly beneficial to patients with additional risk factors for prosthetic joint infection or post-surgical infection

Main applications in Orthopaedics and Traumatology:

  • Coating for primary joint replacement implants, revision surgery, or portions of joint implants;
  • Coating for internal fixation devices (plates, pins, screws, etc.) for fractures,  osteotomy, or neoarthrosis

REFERENCES:

  1. Romanò CL, Drago L, Giavaresi G. et al. Prevenzione delle infezioni peri-protesiche mediante rivestimento riassorbibile anti-batterico: un nuovo approccio? Lo Scalpello 2013;27:88-94
  2. Giavaresi G, Meani E, Sartori M, Ferrari A, Bellini D, Sacchetta AC, Meraner J, Sambri A, Vocale C, Sambri V, Fini M, Romanò CL. (2013) Efficacy of antibacterial-loaded coating in an in vivo model of acutely highly contaminated implant. International Orthoapedics, 2013 Dec 22. doi 10.1007/s00264-013-2237-2
  3. Christensen GD, Simpson WA, Younger JJ. et al. Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices. J Clin Microbiology 1985;22:996-1006