ARTÍCULOS ORIGINALES
Use of human fascia lata in rat calvarial bone defects
Mariano A.R. Amer1, Pablo A. Rodríguez2, Sandra J. Renou3, María B. Guglielmotti3,4
1 Department of Anatomy, School of Dentistry, University of Buenos Aires, Argentina.
2 Department of Endodontics, School of Dentistry, University of Buenos Aires, Argentina.
3 Department of Oral Pathology, School of Dentistry, University of Buenos Aires, Argentina.
4 National Research Council (CONICET), Argentina.
CORRESPONDENCE Dr. Mariano Axel Ramon Amer. Department of Anatomy. School of Dentistry. University of Buenos Aires. Marcelo T. de Alvear 2142 1B, (C1122AAH), Buenos Aires, Argentina. e-mail: marianoamer@gmail.com
ABSTRACT
Tooth loss leads to a decrease in alveolar bone volume, and consequently to the need for guided bone regeneration (GBR) techniques to restore bone anatomy, and the adequate choice of therapy. Fascia lata membrane (FLM) has been used in surgical procedures in neurology, orthopedics, otorhinolaryngology, cardiology, vascular surgery, gynecology, and dentistry for guided tissue regeneration. The aim of the present preliminary study was to evaluate bone tissue response in rat calvarial bone defects covered with human fascia lata membrane (FLM). Eight Wistar rats, 230g body weight, were subjected to bone surgery to create a 5x5mm long/ 1mm deep calvarial bone defect on either side of the median suture, using a piezoelectric scalpel and irrigation. The animals were treated according to the following protocol: Group I (GI): placement of a single layer of FLM (Biotar, Rosario, Prov. de Santa Fe, Argentina) to cover the defects; Group II (GII): double layer of FLM to cover the defects; Group III: no membrane; Group IV: control. All the animals were euthanized 60 days post-surgery; the heads were resected, radiographed, decalcified, and processed for embedding in paraffin and Hematoxylin-Eosin and Masson’s trichrome staining. All bone defects covered with a single or double layer of FLM showed adequate osteogenesis, and none exhibited an inflammatory response. Groups III and IV Control showed scant osteogenesis and no alterations in soft tissues. The results obtained with this experimental model show biocompatibility of FML with the surrounding tissues at the studied time points. No alterations were observed in osteocytic lacunae or osteocytes in the bone after osteotomy using a piezoelectric scalpel. Further studies need to be conducted to assess bone tissue response to FLM in combination with bone substitutes.
Key words: Fascia lata; Skull; Bone regeneration; Piezosurgery; Rats.
RESUMEN
Fascia lata humana en defectos óseos de calota de rata
La perdida de piezas dentarias conlleva la disminucion volumetrica del hueso alveolar y la necesidad de recurrir a tecnicas de regeneracion osea guiada (ROG) para restablecer las condiciones anatomicas y aplicar las terapeuticas adecuadas. La membrana de fascia lata (MFL) ha sido utilizada en intervenciones quirurgicas del area neurologica, ortopedica, otorrinolaringologica, cardiologica, vascular, ginecologica y odontologica para regeneracion tisular guiada. El objetivo de este trabajo preliminar fue evaluar la respuesta tisular de defectos oseos en calota de rata recubiertos con MFL. Se utilizaron 8 ratas Wistar de 230 gr de peso, a las que se les realizo en la calota 2 defectos oseos de 5 x 5 mm de lado por 1 mm de profundidad, con bisturi piezoelectrico e irrigacion, a ambos lados de la linea media, segun tecnica estandarizada. Se realizo el siguiente protocolo: Grupo I (G I): colocacion de una sola capa de MFL (Biotar, Rosario, Prov. de Santa Fe, Argentina) para cubrir el defecto; Grupo II (G II): colocacion de MFL en doble capa para cubrir el defecto; Grupo III (G III): sin membrana; Grupo IV (G IV): control. Se les provoco la eutanasia a los 60 dias. Las calotas fueron resecadas, radiografiadas y procesadas, previa descalcifica - cion, para su inclusion en parafina y coloracion con Hematoxilina-Eosina y Tricromico de Masson. En todos los defectos oseos recubiertos con MFL simple o doble se evidencio una adecuada osteogenesis y ausencia de respuesta inflamatoria y macrofagos. El G III y el G Sham evidenciaron escasa osteogenesis y no mostraron alteraciones en el tejido blando. La MFL en el modelo experimental utilizado evidencio una respuesta compatible con el tejido circundante en los periodos estudiados. El tejido oseo remanente a la osteotomia con bisturi piezoelectrico presento las lagunas osteociticas ocupadas con osteocitos y sin alteraciones. En estudios futuros se evaluara la respuesta tisular con MFL y utilizando un sustituto oseo.
Palabras clave: Fascia lata; Calota; Regeneracion osea; Piezo-cirugia; Ratas.
INTRODUCTION
The use of autologous bone, and heterologous and
alloplastic bone substitutes is widespread in surgery
rooms and dental offices at present. According to
estimates, 2.2 million bone grafts were placed in
2013, including procedures to repair defects in
maxillofacial surgery, neurosurgery, orthopedics, and
dentistry1. Nevertheless, autogenous bone harvesting is associated with complications such as hematoma
and infection, a second surgical site where the graft
is harvested, and potential damage to neighboring
anatomic structures3. Interestingly, the use of
a heterologous or alloplastic graft avoids the
consequences associated with autologous bone
harvesting, as regards morbidity of the donor site and
the limited volume of bone available for grafting.
Barrier membranes used in guided tissue regeneration
procedures can be either resorbable or non-resorbable.
The membrane forms a barrier that prevents migration
of soft tissue into the graft site, and thus enhances bone
healing3. Fascia lata membranes (FLM) have been
used as a resorbable biomaterial4 in a number of
surgical specialties, including dentistry. The structure
of the membrane fibers, its ease of harvesting, and
clinical manageability have made FLM a predictable
biomaterial, which has been successfully used in
numerous surgical fields.
Traditionally, experimental models of bone defects
involved the use of conventional rotating burs to
create the defect. However, these traditional methods
pose disadvantages, such as overheating and damage
to bone tissue. Piezosurgery avoids the complications
associated with conventional rotary drilling methods,
and has become a method of choice for cutting bone
in a number of procedures, including maxillary sinus
floor augmentation, crestal bone splitting, autogenous
bone harvesting, and orthognathic procedures 5.
Clinical and preclinical studies combined with in
vitro studies have shown that piezosurgery produces
clean and precise osteotomies, and decreases
bleeding6. Studies evaluating micromorphological
differences after using a rotary tool, an oscillating
saw, and piezoelectric osteotomy, showed that
piezosurgery preserved the original structure of the
bone7, resulting in a lower number of inflammatory
cells and lower expression of pro-inflammatory
cytokines, 7 and 14 days after surgery8.
The aim of the present study was to perform a
qualitative evaluation of bone tissue response in rat
calvarial bone defects made using a piezoelectric
device and treated with guided bone regeneration with
and without placement of FLM, using histological
studies.
MATERIALS AND METHODS
Eight adult male Wistar rats, 230 g body weight, were
randomly divided into four groups. The animals were
housed in galvanized wire cages, four animals per
cage, at 22°C to 24°C temperature and 52% to 56%
humidity, under 12 hour-light/dark cycles.
All experiments were performed following the
Guide for the Care and Use of Laboratory Animals
of the National Research Council (US)9, and the
protocols were approved by the Ethics Committee
of the School of Dentistry of the University of
Buenos Aires.
On day 0, all the animals were anesthetized by
intraperitoneal injection of 2% xylazine hydroch loride
(5mg/kg body weight) and 5% ketamine
hydrochloride (50 mg/kg body weight). The surgical
area was infiltrated with 0.2 ml lidocaine (1:200.000).
Using a scalpel (Bard-Parker scalpel, 15C blade,
Swam Norton, England), a 15 mm sagittal incision
was made in the parietal region, from the frontal bone
to the occipital protuberance. The calvarium was
exposed by blunt dissection with a Molt curette with
atraumatic non-sharp edges (Hu-Friedy, USA). Bone
defects measuring 5x5mm and 1mm deep were
performed on both sides of the median suture,
avoiding perforation of the inner bone plate.
Bone surgery was performed using a piezosurgery
device (Variosurg, NSK, NAKANISHI INC., Japan)
and a SG3 scaler (NSK,
NAKANISHI INC., Japan).
Animals in Group I (GI)
underwent surgery and
placement of a single
layer of human fascia lata
membrane (FLM) (Biotar,
Rosario, Prov. de Santa
Fe, Argentina) on both
defects (Fig. 1). Those in
Group II (GII) were
subjected to surgery and
placement of a double layer of FLM on both defects. Animals in Group III
underwent surgery without placement of the barrier
membrane, and Group IV served as control.
Fig. 1: Fascia Lata Membrane; a: Macroscopic appearance of FLM; b: Light microscopy of
FLM stained with Masson’s Trichrome. Note the fibrous tissue. Orig. Mag. X 2.5.
The wound was closed with single knot sutures using 5-0 nylon (Ethicon.USA). The rats were maintained on standard chow and tap water ad libitum, and were administered antibiotic therapy and a daily subcutaneous dose of 2 mm3 of Dipenisol. In addition, they received a 1ml dose of Dexamethazone the first two days post-surgery. All the animals were euthanized at 60 days; the heads were resected and fixed in 10% formalin solution. The skulls were radiographed, decalcified in formic acid, and processed for embedding in paraffin. Cross-sections oriented sagittally to the bone defect were obtained and stained with H-E and Masson’s Trichrome.
RESULTS
None of the bone defects covered with a single (G I) (Fig. 2) or double (G II) (Fig. 3) layer of FLM showed an inflammatory response or macrophages, and all exhibited adequate osteogenesis with lamellar bone formation.
Fig. 2: Microphotograph of a histological section corresponding to Group I (60 days post surgery). The defect was covered with a
single layer of human fascia lata membrane. Note the presence of lamellar bone tissue in the bone defect. Also note that the bone of the
surgical bed is of adequate quality, and contains an adequate number of osteocytes. Orig. Mag. X 2.5; Masson’s Trichrome stain.
Fig. 3: Microphotographs of histological sections corresponding to Groups I and II (60 days post surgery). The defects were
covered with a single layer of human fascia lata membrane, and G II defects were covered with a double layer of human fascia
lata membrane. Both groups showed newly formed bone tissue in the bone defect. The fascia lata membranes exhibited no
alterations in their structure, which consisted mainly of a collagen matrix. Orig. Mag. X 50; H-E stain.
It is of note that no alterations were observed in the osteocyte lacunae containing osteocytes or in the surgical bed, where the bone defect was created by osteotomy using a piezoelectric scalpel. No alterations and scant osteogenesis were observed in G III and G IV (Fig. 4). The fascia lata membranes showed no evidence of alterations in their structure, which consisted mainly of a collagen matrix. No sign of inflammation and no macrophages or giant multinucleated cells were observed at the periphery of the membranes.
Fig. 4: Microphotographs of histological sections corresponding to Groups
III and IV (60 days post surgery). No membrane was placed in G III defects,
and GIV served as control. No alterations and scant osteogenesis were
observed in both these groups. Orig. Mag. X 50; H-E stain.
DISCUSSION
Treatment of bone defects created by trauma,
infection, tumor resection, and skeletal abnormalities
poses a great challenge4. Although there are numerous
methods for bone reconstruction, each has specific
indications and limitations. Well established methods
include bone grafting and placement of bone
substitutes or growth factors, among others 10-17.
The concept of GBR has been used in reconstructive
surgery since the mid-1950s.
In 1964, P.J. Boyne published a study on the
regeneration of alveolar bone beneath cellulose
acetate filter implants18.
As stated by Retzepi M et al (2010)19,
bioresorbable membranes were developed
to avoid the need for surgical removal.
The results of the present study showed
adequate wound healing of bone defects
treated with a single or double layer of
human fascia lata membrane (FLM; Biotar,
Rosario, Prov. de Santa Fe, Argentina)
(Group I and Group II respectively), as
shown by the finding of osteogenesis with
lamellar bone formation. The defects in
Group III, which were not treated with
the FLM, exhibited less lamellar bone
at the surgical defect. Attention must be
drawn to the biocompatibility of FLM, as
evidenced by the absence of inflammation,
macrophages, and giant multinucleated
cells.
The bone of the surgical bed created by
osteotomy using a piezoelectric scalpel
showed lamellar bone tissue of adequate quality,
and contained an adequate number of osteocytes,
demonstrating that piezosurgery does not alter the
structure of the bone at the surgical site.
The results obtained with the experimental
model used here, involving osteotomy with a
piezoelectric scalpel to create a bone defect in rat
calvaria and treatment of the defect with GBR using
FLM, showed adequate osteogenic repair and
adequate quality of the bone at the surgical site,
both when using a single layer and a double layer
of FLM.
CONCLUSION
Tissue response to FLM observed 60 days postsurgery using the experimental model presented here, provides further evidence of the biocompatibility of human fascia lata, as well as of its enhancing effect on osteogenesis during the repair process of experimentally created bone defects. Further studies need to be conducted to assess bone tissue response to FLM in combination with bone substitutes.
ACKNOWLEDGEMENTS
The authors acknowledge the collaboration of Marcela Alvarez and Ricardo Orzusa, technical assistants at the Departments of Oral Pathology and Biochemistry respectively, of the School of Dentistry, University of Buenos Aires, Argentina. This work was supported by research Grants UBACYT 2014- 2017 20020130100332 and PIP CONICET 2014-2016 11220130100091.
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