Keywords
histology - periodontal regeneration - platelet-rich plasma
Introduction
The alveolar bone, periodontal ligament (PDL), and cementum are the integral structural
components invariably subject to change during periodontal disease. Open-flap debridement
promotes the growth of the microorganism community due to long junctional epithelium
formation, leading to the less stable attachment.[1]
Bone replacement grafts are in use for a long time, to enhance bone formation and
regeneration of periodontal tissues in vertical osseous defects. Bone formation occurs
by processes such as osteoconduction, osteoinduction, or osteogenesis.[2] Autogenous bone graft remains the gold standard,[3] but postoperative morbidity and limited available volume of intraoral autogenous
bone are the major drawbacks to its extensive use.
Hydroxyapatite (HA), a synthetic alloplastic bone graft, is chiefly utilized in the
treatment of osseous defects. The porous form of HA forms through a hydrothermal conversion
of the calcium carbonate of coral exoskeleton. The HA forms a scaffold to support
the fibrovascular ingrowth with subsequent bone formation by osteoconduction.[4]
The lack of osteoinductive potential is a disadvantage for porous 10-1055-s-0040-1714651_00084_
(PHA) usage in intrabony defects.[5] Histological evidence of lamellar bone formation, the significant component within
the pores, has been found in defects treated with PHA.[4]
[6] However, other reports suggest encapsulation of graft material within the forming
of new bone, without any evidence of its resorption.[5]
[7]
Newer regeneration therapies have focused on the biomimetic approach, utilizing naturally
occurring biological modifiers such as polypeptide growth factors (GFs) to accelerate
wound healing. The GFs regulate several other cellular events in tissue regeneration
involving chemotaxis, cell proliferation, differentiation, or matrix synthesis via
binding to specific cell surface receptors.[8]
Recombinant GFs either singly or in combination have provided evidence for bone regeneration
in periodontal defects in both animal and human trials.[9]
[10]
[11]
[12]
[13]
[14]
[15] However, these are cost-prohibitive, thus not used clinically to a great extent.
Alternatively, platelet-rich plasma (PRP), procured from whole blood is a rich source
of autologous GFs, and can promote periodontal regeneration. These GFs are approximately
300 times more than that of the levels present in normal plasma. Although plasma is
considered to have more than 30 different GFs, platelet-derived growth factor (PDGF)
and transforming growth factor-β (TGF-β) present in platelets are the most important.
They assist in tissue healing, mainly by stimulating proliferation and differentiation
of mesenchymal cells.[16]
[17] PRP with synthetic porous HA triggers the formation of new bone in the healing site
by the release of GFs. The osteoinductive and osteoconductive functions of the HA
stimulate the differentiation of osteoblastic/progenitor cells.
Histological evidence of new attachment in humans is sparse. Nevins et al were the
first to demonstrate periodontal regeneration, including bone, PDL, and the cementum
in human histological specimens of severe osseous defects following treatment with
recombinant PDGF.[18]
This study aimed to obtain histologic evidence, if any, of new attachment in humans,
following treatment of osseous defects with HA and PRP mix.
Materials and Methods
Patient Selection
Following an ethical committee consent, five adult patients 24–53 years of age were
enrolled in the study. Patients presenting clinically with probing pocket depth of
5–8 mm and a clinical attachment level greater than 5 mm, and with radiographic evidence
of at least one advanced osseous defect involving the distobuccal root up to the apical
third with a vertical component of at least 5 mm were selected. For the teeth associated
with the osseous defects, a planning of radisection of the distobuccal root by two
independent periodontists ([Fig. 1]) was done. Patients who were systemically healthy and with no contraindication to
periodontal therapy were included. Patients with systemic diseases and tooth with
osseous defects with good regenerative potential were excluded from the study.
Fig. 1 (A) Preoperative clinical view. (B) Intraoperative view of defect. (C) Defect filled with platelet-rich plasma and porous 10-1055-s-0040-1714651_00084_.
(D) Flap closure by means of interrupted sutures.
Presurgical Therapy
Preoperative hematological assessment including a complete blood count was done. Initial
therapy consisted of full-mouth scaling and root planning utilizing both hand and
ultrasonic instruments under local anesthesia. Oral hygiene instructions were given
at each visit and were reinforced throughout the study. They were repeated until the
patient achieved a full-mouth plaque score of less than 25% and there was absence
of clinical signs of gingival inflammation. Symptoms of trauma of occlusion, if detected,
were corrected.
Preparation of Platelet-Rich Plasma
PRP was procured by the method previously described by Sonnleitner et al.[17]
Briefly, PRP was extracted 30 minutes prior to surgery using venipuncture. Further,
20 mL of blood was drawn from each patient by venipuncture of the antecubital vein
in the forearm into a 20 mL syringe. Then, 10 mL of blood was collected into two glass
tubes containing 10% trisodium citrate solution as an anticoagulant. The glass tubes
containing blood were centrifuged at 1,200 rpm for 20 minutes, resulting in the separation
of two fractions: plasma at the top and red blood cells at the bottom. The plasma,
along with the top 2 mL of red blood cells, was aspirated with the help of “Eppendorf
pipettes.” This fraction was again centrifuged at 2,000 rpm for 15 minutes to get
three basic fractions: platelet-poor plasma (PPP) at the top of the preparation (supernatant),
PRP in the middle, and red blood cell fraction at the bottom. The top 80% corresponding
to PPP was aspirated with a pipette, leaving the residual (0.5–2 mL) platelet concentrate.
Surgical Protocol
The surgical procedure was performed under local anesthesia. Buccal and lingual sulcular
incisions were used and a mucoperiosteal flap was elevated. Care was taken to preserve
as much interproximal soft tissue as possible. Complete debridement of the defects
as well as scaling and root planning were achieved with the use of an ultrasonic device
and hand instruments ([Fig. 2]).
Fig. 2 (A) Five-month postoperative clinical view. (B) Reentry of the treated site. (C) Distobuccal root removed, together with some surrounding hard tissue. (D) Flap closure by means of interrupted sutures following radisection.
Intrabony defects after soft tissue debridement were measured vertically from the
base and the crest of the defect to the cemento-enamel junction.
Treatment of the Defect
The bone graft was a synthetic, osteoconductive, nonceramic form of HA (Osteogen -
Impladent, New York, United States) with the PRP in a proportion of 1:1. Coagulation
of PRP/synthetic HA mixture was prepared with 5 μmL of 10% calcium chloride, which
takes up a sticky gel consistency. This mixture was then packed into the bony defects
to the bony crest level ([Fig. 2C]).
Flaps were sutured using interrupted sutures ([Fig. 2D]). Prescription of antibiotics (Amoxicillin 1.5 g every 6 h for 5 d) and 0.12% chlorhexidine
gluconate rinse (every 12 h for 2 weeks) along with oral analgesic (Ibuprofen 400
mg every 8 h as necessary) was given.
Postoperative Care
Periodical assessment was done weekly for the 1st month, every other week for the
2nd month, and monthly until biopsy. Suture removal was done after 2 weeks postoperatively
cleansed with 0.12% chlorhexidine gluconate on a cotton swab. Patients were given
guidelines for brushing at the end of the second postoperative week. They were examined
weekly up to 1 month after surgery and then in the 3rd and 5th months. Postoperative
precautions with care included the emphasis on oral hygiene and mechanical plaque
removal, whenever mandatory. Radiographs were taken at regular intervals to check
for bone formation ([Fig. 3]).
Fig. 3 Intraoral periapical radiographs taken at different time intervals.
Radisection
All biopsies were sent to the laboratory for analysis. Radisection of the vital tooth
was performed at 1st, 3rd, and 5th months in different patients ([Fig. 3C]) and the specimens fixed in 10% neutral formalin and demineralized in 20% formic
acid over a period of 2 months and embedded in paraffin wax. Sections 5μm thick were
cut through each biopsy and were stained with hematoxylin and eosin. Stained sections
were photographed under light microscope attached to a Motic (version plus 3.0). Histologic
evaluations of the specimens were conducted by an oral pathologist.
The patients were asked to report after 10 days to remove the sutures and to debride
the wound. After healing of the surgical sites, patients were advised to report for
reviews at regular intervals to check for wound healing and vitality of the teeth.
Results
All five patients completed treatment and experienced no adverse reactions related
to treatment. Clinically, wound healing appeared enhanced in PRP/PHA treated sites.
Following surgery, the distobuccal roots were extracted at 1, 3, and 5 months in different
patients and the specimens obtained were processed and examined under light microscope.
Histological evaluation was performed for four of the five specimens as one specimen
was not evaluable due to difficulties encountered during processing.
The photomicrograph of specimens taken 1 month after grafting showed no evidence of
particulate graft material. Encapsulation of graft with a fibrous tissue was also
not observed. Inflammatory cell presence was sparse, with no characteristic. Mature
collagen was observed in the treated area. Immature bone formation was also observed
([Fig. 4A]).
Fig. 4 (A) Photomicrograph of specimens taken 1 month after treatment shows immature bone with
mature collagen. Few inflammatory cells are seen (hematoxylin-eosin stain magnification
4×). (B) Photomicrograph of specimens taken 3 months post operation shows mature bone formation
(hematoxylin-eosin stain, original magnification 4×). (C) Photomicrograph of specimens taken at 5 months shows root bit along with presence
of cellular connective tissue and mature bone (hematoxylin-eosin stain, magnification
4×). (D) Photomicrograph of higher magnification of the specimens taken at 5 months shows
mature bone with osteocytes within lacunae and presence of resting lines (hematoxylin-eosin
stain, magnification 10×). Note: CT, connective tissue; D, dentin; C, cementum; B,
mature bone; Arrow, resting lines.
The photomicrograph of specimens taken at 3 months after grafting showed a small amount
of mineralized bone with fibrous connective tissue ([Fig. 4B]).
The photomicrograph of specimens taken at 5 months showed mature bone with osteocytes
within lacunae along with the presence of resting lines. There was also the presence
of cellular connective tissue ([Fig. 4C ]
[D]). None of the slides showed PDL or acellular cementum.
Discussion
The limitations of traditional therapies have promoted the development of tissue engineering.
This emerging field is concerned with the development of natural biological surrogates
that restore, maintain, or improve tissue structure and function. Three general strategies
have emerged for the engineering of tissues. The first is a conductive approach in
which synthetic scaffold materials amenable to infiltration of specific cell types
are implanted into a site of disease or defect. The materials provoke the conduction
of desired cell types while blocking the conduction of unwanted cell types. The second
approach involves the inclusion of bioactive factors (e.g., GFs) into the aforementioned
synthetic scaffolds. They are chosen to spur the infiltration of the specific cell
types and induce the formation of any particular type of tissue. The third approach
is on the basis of seeding scaffolds with cells in vitro, followed by implantation
of the cell construct.[19]
Basic and clinical research has focused on the application of GFs for the regeneration
of tissues. This can be achieved through gene therapy. In a review, Yao and Eriksson
reported that short shelf life and inefficient delivery to target cells are the major
concerns associated with local administration of recombinant human GFs. The GFs were
expensive and sufficient doses were required to achieve any obvious therapeutic effect.[20]
Another easy, cost-effective way to obtain concentrations of GFs for tissue healing
and regeneration may be autologous platelet storage via PRP.
GFs such as TGF-β and PDGF determine the rationalization of the use of PRP as a bone
regenerative stimulating agent. These are carried into the regenerating site with
an ideal carrier, like the patient’s platelets.[8]
The ability of PRP to enhance the consolidation of bone graft has been well established
since 1998 by the pioneering works of Marx et al.[21]
[22]
Several studies have exemplified the role of platelet formulations in regeneration
of soft/hard tissues, including formation of new bone. In these studies, different
types of bone replacement grafts such as demineralized bone powder, Bio-bone/Bio-Oss,
HA, and other forms of allografts have been used in combination with PRP gel in the
treatment of osseous defects.[23]
[24]
During the bone regeneration process, the GFs present in the PRP gel carry out important
functions for the initiation and maintenance of the proliferation, and differentiation
of the osteoblastic precursor cells and osteoblasts themselves, leading to bone formation.[22]
In this study, the PRP was prepared using a method previously described, except that
thrombin was not used to avoid any risk to the patient by mixing PRP with any component
of animal or human origin.[25]
[26]
Nevins et al[18] demonstrated periodontal regeneration including bone PDL and cementum after 9 months
in sites treated with recombinant human PDGF. However, the dynamics of the healing
process were not studied as all the specimens were obtained after 9 months. To gain
further insight into the regenerative process, the specimens in this study were obtained
at 1, 3, and 5 months of intervals.
In our study, the photomicrograph of a specimen taken at 1 month after grafting showed
the presence of osteoid formation, with mature collagen and sparse inflammatory cells.
There was no evidence of fibrous encapsulation of the graft or remnants of graft material.
Previous histologic studies have showed that though the use of porous synthetic HA
resulted in bone formation, there was limited osteogenesis with the graft particles
being encapsulated in the new bone formed.[4]
[5]
[7]
[27]
This early resorption of PHA particles in this study could be the result of increased
inflammatory cell infiltrate following PRP application, as suggested by Hartwig et
al.[28] Stimulated platelets actively synthesize proinflammatory cytokines (e.g., CD40L,
IL-β) and activate macrophages by the release of chemokines. The increased number
of macrophages may have accelerated the resorption of the bone graft.
However, at the 3rd-month stage, this inflammatory cell exudate did not translate
into excessive osteoclastic activity. The presence of collagen and bone formation
at the healing site is suggestive of more osteoblastic than osteoclastic activity.
Kubota et al[29] in an in-vivo study have shown osteoclastic activation by PDGF-BB released in an
inflammatory environment. However, this in-vivo result may not be truly reflective
of the in-vitro healing events in a vertical osseous defect.
The subsequent slides have shown the maturation of the newly formed bone, and, at
the 5th month, definite evidence of resting lines in the newly formed bone with osteocytes
in the matrix suggests bone formation by osteoblastic activity.
We may summarize the results as follows:
Current opinion about the use of PRP in regenerative procedures is not complimentary
to the PRP gel. We propose that the positive results obtained in this study could
be the result of the following:
-
Periodontal defects being much smaller in size than oral surgical defects consume
much less PRP gel. Thus PDGF-BB, which is the major component of PRP with osteoclastic
activity, may be in smaller concentrations. PDGF-BB has also been shown to have a
very short half-life[30] and hence, the osteoclastic activation may not be of much clinical significance.
-
When mixed with a slow resorbing alloplast like PHA, the action of PRP may be prolonged
for an interval sufficient enough to release PDGF and TGF-β and hence induce osteoblastic
activation.
-
The early evidence of bone formation at the 3rd month itself is clear evidence of
bone formation as a result of PRP and not just the PHA alloplast.
In this study, there was no evidence of PDL formation as shown by Nevins et al[18] probably because:
-
The PHA mixed with PRP may have precluded the possibility of the fibrin glue adhering
to the root and hence may not have impeded apical migration of junctional epithelium.
-
The experimental sites were not ideal conditions for regeneration due to their extensive
bone loss and gingival recession.
It may thus be a fair assumption to state that in defects with better prognosis, the
regenerative potential would only increase. A more precise understanding of the dynamics
of GF release from PRP used in smaller periodontal defects versus more extensive surgical
wounds would help to clarify their exact role in osteoblast/osteoclast activation.
The evidence of this study would seem to indicate that some of the criticism about
PRP in periodontal defect treatment is unwarranted.
In conclusion, platelet-rich fibrin in conjunction with osteoconductive materials
prove to accelerate bone formation in vertical osseous defects.
