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ArticlesThe application of PRGF-ENDORET

The application of PRGF-ENDORET

Treatment methods development for regeneration acceleration of soft tissues and socket bones is very important for delayed treatment protocols for short, medium or prolonged time aiming to shorten expectation time for patient and improve regenerated tissue quality. Post-extraction socket filling with PRGF®-ENDORET® with its further densification with autogenous fibrin allows us to obtain sufficient socket bone quantity (density more than 500 Hounsfield units) of sufficient quality (bone of type I, II and III) to secure primary implants stability, both in internal and external socket walls, for the period of 4 – 8 weeks. Socket treatment after extraction with PRGF®-ENDORET® and autogenous fibrin is simple, economic and predictable biotechnological process for socket bone and keratinized gum regeneration that allows us to considerably shorten patient expectation time without any adverse effects, as show many years of experience.

 

Introduction

During the last ten years dentistry has made considerable progress due to more profound knowledge of dental pathologies and development of new biological techniques and protocols. We may look back into the past only for a moment to understand that the protocols which were indisputable some time ago – as for example, implantological treatment by Branemark – are completely substituted today. This standard original procedure meant a 12-month healing period before implant installation after tooth extraction, and the patient had to wait for 3-6 months before the next surgery. Now we use other criteria to determine time period between tooth extraction (exodontia) and implantation. According to modern classification there is an immediate implant installation and delayed implant installation – for a short, medium or prolonged period – depending on the period when the implant is placed – immediately upon extraction or after 6-8 weeks, 3 months and 9 months.

Certainly, that immediate implant installation has many functional and aesthetic advantages for a patient, apart from considerable treatment time shortening. Nevertheless, at certain circumstances to guarantee functional and esthetical success of implantation the complete regeneration of post-extraction socket and keratinized gum are needed. Certain situations, such as space-occupying periapical lesions, three-wall defects, large craters around teeth or gum recession may be contraindications for immediate installation of implants. In such cases for socket healing and regeneration longer time is needed. On the other hand, treatment delay after teeth loss increases risk of socket resorption, loss of bone height and dental arch thickness which in certain cases may make implant installation more complicated or absolutely impossible. It is necessary to create a protocol which would accelerate the process of soft tissues and alveolar bone regeneration to obtain quantity and quality of keratinized tissues and bones ideal for implantation, and which at the same time reduces patient expectation time.

Post-extraction socket filling with preparation, enriched with growth factors (PRGF®-ENDORET®) (1) is a biotechnological alternative for alveolar bone regeneration acceleration.

PRGF®-ENDORET® consists of small volume of high-specific preparation from blood plasma, enriched with platelets, which is obtained rapidly and prepared simply from patient own blood. Its activation produces three-dimensional biocompatible fibrin matrix. As a result, several proteins and growth factors are secreted which promote bone healing acceleration and bone regeneration (8).

 PRGF®-Endoret® - is 100% autogenous and biocompatible product. Today it is more correct to talk about PRGF®- Endoret® technology, because it means not a single product, but several compositions with therapeutic activity that are obtained in a simple way from patient own blood by single preparation protocol:

Supernatant PRGF®-Endoret® is an excellent nutritious medium for stem cells and autogenous bones. It contains growth factors and plasma proteins obtained during clot retraction PRGF®-Endoret® (Figure 1).

Activated liquid PRGF®-Endoret®: PRGF®-Endoret® activation by activator PRGF®-Endoret® (calcium chloride) promotes release of proteins and platelet growth factors which leads to saturation with signals that may create bioactive surface on teeth implants to accelerate osteointegration. This liquid may also be used for infiltration of muscles, tendons, skin, joints, wounds, etc. (Figure 2).

Clot PRGF®-Endoret®: in 3-5 minutes activated liquid PRGF®-Endoret® turns into a three-dimensional fibrin matrix impregnated with growth factors which may be used for many procedures, from socket regeneration after tooth extraction to treatment of musculoskeletal and vascular pathologies (Figure 3).

Autogenous fibrin: clot retraction, obtained from fraction 1 PRGF®-Endoret® allows obtaining dense, elastic and completely biocompatible fibrin that may be used in many cases as isolating membrane (Figure 4).

Thus, this article describes the technique, used in 11 patients of an average age over 50 years. This patient group was selected to observe effects of PRGF®-Endoret® and autogenous fibrin. The aim of the study was to confirm growth factor efficiency in combination with fibrin matrix, along with decrease of expectation time, necessary for adequate bone healing and regeneration. The study also assessed autogenous fibrin potential as biocompatible material for optimal socket filling. To achieve this with the help of Cone Beam Computerized Tomography we determined density and quality of alveolar bone on 8-13 weeks after extraction with further results processing with software BTI Scan® II(3).

 

PRGF*-Endoret* supernatant, enriched with growth factors

Figure 01: PRGF*-Endoret* supernatant, enriched with growth factors Figure 02: Liquid PRGF*-Endorel" secures bioactivation of dental implants surface to improve their osteointegration.

 Elastic properties of autogenous fibrin

Figure 03: Clot PPGF-Endoret - is three-dimensional matrix from fibrin and other components, impregnated with growth factors. Figure 04: Elastic properties of autogenous fibrin.

Preparation

Total 11 patients (4 males and 7 females) aged 45 - 71 years (mean age 53.8 years) were properly informed and made an experimental group. PRGF®-ENDORET® was prepared from small blood volume (20 cm3), taken from peripheral vein using sodium citrate as anticoagulant (citrated tubes of PRGF®) (Figures 5 - 10). The tubes later were centrifuged to divide plasma and erythrocytes (centrifuge BTI IV). Plasma layer was determined according to protocol PRGF®-Endoret®::

Equipment of PRGF*-ENDORET* system

Figure 05: Equipment of PRGF*-ENDORET* system. Figure 06: Citrated PRGF for blood sampling.

Various fractions in 9-ml tube after centrifugation

Figure 07: After centrifugation plasma and blood of different colours may be observed. Tubes BTI PRGF*-ENDORET* have calibrating scale to see the obtained plasma volume, because it varies depending on patient hematocrit. Figure 08: Various fractions in 9-ml tube after centrifugation.

Plasma mixing device (PTD)

Figure 09: Plasma mixing device (PTD). Figure 10: PTD in position for fractions separation.

 

Fraction 1: This is the upper part of the total plasma, obtained after centrifugation. This fraction has the same platelet concentration as circulating blood. Its volume is calculated as minus 2 ml of plasma layer over leucocytes film.

Fraction 2: This is 2 ml of plasma layer over leucocytes film. It contains 2.5 times more platelets than circulating blood. It is very important during the fraction sampling not to mix it with leucocytes that produce tissues inflammation.

After these two fractions separation each may be used in different situations. The upper fraction (fraction 1) is used as autogenous fibrin membrane for thickening of socket crown portion which would provide support for growth of bones and soft tissues.

After obtaining necessary plasma fractions their activation is carried out that produces clot or membrane formation. To achieve this we add 50 mcl (0.05 cm3) of PRGF®-Endoret® activator for 1 cm3 of plasma. Activator quantity is measured with insulin syringe, calibrated for mcl, and then activator added to plasma. Clot is formed in 5-8 minutes. This time varies in inverse proportion to platelets number.

Thus, the more platelets we have, the less time aggregation would take.

These data are important due to individual variation in platelets blood number, with physiological range 150 000 – 400 000. If we store activated plasma at body temperature (37°С), this time is shortened and clot formation would be more controllable.

The procedure aimed at socket regeneration with PRGF®-Endoret® is performed as one surgical procedure together with extraction. For this we place into socket coagulated clot PRGF®-ENDORET®, obtained from fraction 2, and then we thicken the socket with retracted fibrin membrane that has excellent elastic and homeostatic properties.

Apart from barrier effect, leaving the space for fibrin thickening, the clot reabsorbs completely by osteoconductive material during 3-4 months (Figures 11 - 13).

Newly formed fibrin clot 8 minutes after activation

Figure 11: Newly formed fibrin clot 8 minutes after activation.

 

Fibrin membrane is received 45 minutes after activation immediately after clot retraction

Figure 12: Fibrin membrane is received 45 minutes after activation immediately after clot retraction.

 

Complete fibrin retraction 1 hour after activation

Figure 13: Complete fibrin retraction 1 hour after activation. With this consistence it may even be stitched.

 

For accurate supervision of bone regeneration process the patients underwent CBCT examination during weeks 8-13 after extraction. With the software for images processing and analysis BTI Scan® II it was possible to determine bone density and classify bone quality by Lekholm and Zarb.  In order to obtain these data we determined bone density in three different points in regenerated socket and in several places 0.5 mm each, both at internal (1 mm inside) and external (1 mm outside) perimeter, which afterwards would constitute the future implant site. Therefore, it was possible to determine bone regeneration not only inside the socket, but also, more importantly, the density and quality of socket walls, where the implant would be placed, and on which implant primary stability depends.

 

Results

Immediately after PRGF®-ENDORET® activation the process of growth factors (GF) release is started. These GF would play the key role in bone revascularization and regeneration, rendering mitogenic and proliferative effects on endothelial and osteo-progenitor cells. Aiming at characterization of the content of growth factors in PRGF®-ENDORET® , we measured the quantity of basic growth factors released  from platelets, namely of PGF (platelet growth factor), ß-PGF (ß-transforming), IGF-1 (insulin-like), EGF (epidermal), VEGF (vascular endothelial growth factor), and HGF (hepatocyte growth factor) (Figures 14 and 15).

 

Basic growth factors concentration in PRGF'-ENDORET

 

In PRGF®-ENDORET® the most presented are the first 3 factors, and their effects on healing and bone regeneration is are described in literature in detail.

For example, PTF is known for its ability to increase osteoblasts proliferation in vitro, while ß-PGF in certain dose stimulates synthesis of proteins – collagen derivatives, such as collagen of type I and V, apart from augmentation of matrix mineralization and improvement of implant survival. Finally, IGF-1 stimulates bone formation, inducing cells proliferation, differentiation and biosynthesis of collagen type I. It is also known for its mitogenic function in multinuclear osteoclasts. However, the most important note that according to various studies this GF combination has synergic effect on bone regeneration. Moreover,  PRGF®-ENDORET® protein activation causes release of large quantity of proteins and growth factors playing key role in proper bone regeneration. In this respect we should make accent on angiogenic effects of VEGF, because they are vitally important for appropriate supply of oxygen and nutrients to regenerated tissues. Immediately after preparation of PRGF®-ENDORET® the tooth extraction is conducted and the socket is filled with PRGF®-ENDORET®, and thickened with autogenous fibrin, as it is illustrated by Figures 16-20.

We should note one aspect, namely that the quantity of used PRGF®-ENDORET® varies depending on socket size, to adequately fill the defect formed. 2-3 months after sockets filling and thickening the patient underwent CT; the results were analyzed by software BTI Scan® II. As we mentioned before, this program is excellent diagnostic tool to access bone tissue quantity and quality, which helps securing implantation predictability.

In fact is seems that these two variables have a decisive effect on implantation success or failure, independently of implants place. Failure rate increases when bone quantity is insufficient or its quality is low which would immediately affect implant primary stability.

 

Post-extraction socket scheme

Figure 16: (а) Post-extraction socket scheme. (b) Socket filling with clot PRGF'-ENDORET* and autogenous fibrin membrane obtained by the same technique, (с) Regeneration of keratinized tissues and post-extraction socket after 12 weeks.

 

 In this case suturing, in addition to fibrin proper adhesive properties

Figure 17: We carried out extraction, socket curettage and filling, as described above. In this case suturing, in addition to fibrin proper adhesive properties, stabilizes and holds fibrin membrane. Figure 16: Extraction zone 24 hours later.

Fibrin membrane epithelisation after 15 days and 3 months after

Figure 19: Fibrin membrane epithelisation after 15 days. Epithelisation period varies from 5 to 15 days depending on socket size and patient. Figure 20: Epithelial regeneration seen 3 months after.

For example, bone of type IV which is perceived as low quality bone is characterized by low density of matrix of soft trabecular bone, comparing to type II and III, which quality secures much higher primary implant stability.

BTI Scan® II program allows correlating bone density with bone quality classification by Lekholm and Zarb. Nevertheless, we present a new more accurate classification which includes 5 classes according to average density in Hounsfield units to determine bone type (Figures 21 – 25). During the last several years our research group has made large efforts aimed at thorough study and description of PRGF®-ENDORET® and all its possible therapeutic applications (4-6). This study demonstrates huge potential of PRGF®-ENDORET® as means for socket bone regeneration in patients over 50 years of age, who have decreased osteogenic activity (8).

 

Bone type Histology Usual position Hounsfield units
Type I Very dense cortical bone Frontal zone of inferior jaw > 1400 - 1050
Type II Dense cortical bone (3-4 mm), surrounding dense trabecular bone.

Frontal zone of inferior jaw

Lateral zone of inferior jaw

1000 - 850
Type III Less dense cortical bone (2 mm), surrounding dense trabecular bone. Frontal and lateral zone of inferior and superior jaw 800 - 550
Type IV Very thin cortical bone (0.5 - 1 mm) around low density trabecular bone. Lateral zone of superior and partially inferior jaw 500 - 400
Type V Trabecular bone of very law density Lateral zone of superior jaw 350 - 100


Table 1: Bone density with respect to bone type and the most characteristic zones.

 

It is important to assess density both inside, and along outer perimeter of future implant site

Figure 21: Bone of type I (10S0 - 1400 Hounsfield units). It is important to assess density both inside, and along outer perimeter of future implant site.

 

Bone of type II (850 - 1 ООО Hounsfield units)

Figure 22: Bone of type II (850 - 1 ООО Hounsfield units). Figure 23: Bone of type III (550 - 800 Hounsfield units).

Bone of type IV (400 - 500 Hounsfield units)

Figure 24: Bone of type IV (400 - 500 Hounsfield units). Figure 25: Bone of type V (100 - 350 Hounsfield units).

 

We should note that even 2-3 months after extraction (more exactly, on weeks 8 - 13) results analysis with program BTI Scan® II showed high density and sufficient bone quantity in sockets, filled and thickened with PRGF®-ENDORET® and autogenous fibrin (Figures 26 - 28). Namely, we obtained average density 534 Hounsfield units in socket centre – the zone where 12 weeks before the bone was absent completely, and the density was zero. However, no doubt that the most prominent result was high bone density both in the middle and along the outer perimeter of future implantation zone reaching more than 600 Hounsfield units which guaranteed a high primary stability.

Scheme of three measurements in socket to access bone regeneration quality

Figure 26: Scheme of three measurements in socket to access bone regeneration quality.

 

Density results inside the socket, obtained in average from 3 measurements

Figure 27: Density results inside the socket, obtained in average from 3 measurements in 3 different points in the socket centre. Figure 28: Density measurement was conducted every 0.5 mm both by 1 mm inside and 1 mm outside the hypothetic position of the future implant. This plot presents the picture on weeks 8-13 here after

 

Along with this, the results of bone quality assess for implant installation confirm bone formation of suitable quality for implants: in 6 patients the bone of type II was formed, and in 5 patients – the bone of type III.

ЭThese data confirm the fact that this technology application is a considerable progress for immediate procedure, as well as for a delayed procedure for a short or medium term, because it significantly shortens time between surgeries. It is also necessary to consider that according to traditional literature full healing of post-extraction socket takes 12 months, and admitting that during this time dental arch thickness may decrease by 50%. Therefore, as it was mentioned before, the issue is not only the intra-operative period shortening, but mostly the function, aesthetics and improved prognostic possibility for the future treatment.

The most important aspect is that not a single patient suffered from discomfort, inflammation or infection after extraction and filling with PRGF®-ENDORET®. In our opinion this was due to autogenous fibrin use for socket thickening. Some authors think socket closure is not the primary goal; other authors prefer patch shifting for socket primary closure. However, this technique may decrease gum thickness around implant which disturbs patient aesthetic appearance. In contrast to this, autogenous fibrin does not lead to any side effects, and this procedure is safe and simple for specialist, as well as economic and effective for patient. Notwithstanding the fact that the number of patients in this study was small, we tried to study regeneration effect after PRGF®-ENDORET® application and further thickening with fibrin membrane for various teeth locations – both on the superior and inferior jaws. Our results confirmed that PRGF®-ENDORET® has prominent regenerative effect in various studied sockets, which testifies to its therapeutic potential.

These results implied the following: what if we study the same application protocol for PRGF®-ENDORET® for the new patient group during the longer time? Will the density characteristics increase considerably and the bone density change?

So said, so done. We studied 8 new patients (3 males and 5 females) who were examined by BTI Scan® II on weeks 14-16 after extraction and who demonstrated very similar density parameters inside sockets (567 Hounsfield units) and a little less density, though insignificantly, outside and inside the proposed implantation site (Figures 29 and 30).

This means that we should not seek for favourable regenerative effect enhancement of PRGF®-ENDORET® after more prolonged time, but otherwise, we should imply improvement and patient benefits after shorter time.

Thus, till what time the primary determination of bone density and quality would continue yielding the same positive results? This question arises due to excellent results for some patients, especially who underwent CT on week 8 after extraction. Therefore, we presume there is more expedient to study this aspect to discover, if it is possible to achieve the same positive results for even lesser time, shortening the period of patient expectation.

 

Bone density inside socket and on the periphery of the future implant site

Figures 29 and 30: Bone density inside socket and on the periphery of the future implant site on week 14-16 after extraction.

 

Conclusion

This article discussed socket treatment after extraction applying the technique PRGF®-ENDORET® which had been previously described by our research group (7, 9) as predictable. This is probably the best biological material for socket treatment after extraction because it is 100% autogenous product obtained in simple and economic way (from 20 cm3). In case of severe loss of vestibular plate it may be used in combination with biomaterials or preferably with autogenous bone (7, 9).

In the previous complex clinical study the same procedure was used in more than 200 patients who were followed for 5 years without any side effects. On the contrary: more rapid epithelisation was observed, less pain and less inflammation. No complications were observed, and not a single case of dry alveolar neuralgia. Further studies are needed to compare effectiveness of this procedure with others, such as shifting of connective tissue transplant or split patch. However, soft tissues regeneration is more effective using PRGF®-ENDORET®, and it is recommended for the majority of extractions, no matter if further implant installation is planned or not. As for the time, this study makes obvious that small defects demand 8-10 weeks; for larger defects – in 14-16 weeks better bone quality may be guaranteed. (Figures 31 – 35).

 

aseline X-ray scan of one patient, involved in the study

Figure 31: Baseline X-ray scan of one patient, involved in the study. Lesion in bifurcation zone and apical lesion in medial root are indications for tooth extraction.

 

Photo during extraction operation, and after 3 months

Figure 32: Photo during extraction operation. Figure 33: Photo of obtaining access for implant installation after 3 months.

 

 Photo of implant installation procedure

Figure 34: Photo of implant installation procedure. It is seen that regeneration degree allows for implant installation. Figure 35: X-ray control of final rehabilitation after 3 months.