Clinical Trial Details
— Status: Active, not recruiting
Administrative data
| NCT number |
NCT01997437 |
| Other study ID # |
11.G34.31.0065 |
| Secondary ID |
|
| Status |
Active, not recruiting |
| Phase |
N/A
|
| First received |
November 5, 2013 |
| Last updated |
January 25, 2016 |
| Start date |
December 2013 |
| Est. completion date |
December 2016 |
Study information
| Verified date |
January 2016 |
| Source |
Kuban State Medical University |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
Russia: The Ministry of Education and Science of the Russian FederationRussia: Ministry of Health of the Russian FederationRussia: Ethics Committee |
| Study type |
Interventional
|
Clinical Trial Summary
The proposed protocol will involve the replacement of the trachea using a synthetic
bioengineered scaffold seeded with autologous mononuclear cells as an intraoperative
solution for patients with with benign and malignant laryngo-tracheal diseases or other
terminal conditions of the trachea.
Tracheal transplant is indicated as the only therapeutic alternative in cases where
instrumental, endoscopic and other evaluations show that the length of residual healthy
airways (about 6 cm or longer than 50% of the airway length) and the localization and
extension of the obstruction make it impossible to perform a surgical resection of the
pathological segment.
In addition to tracheal surgical transplant techniques, this protocol requires knowledge and
experience with autologous cell preparation as well as scaffold seeding procedures.
Description:
Before transplantation the patients will have the laboratory and instrumental evaluations.
Three days before the transplantation the patient will be underwent bone marrow aspiration.
The bone marrow mononuclear cells (MNC) will be isolated from the red blood cells (RBC) in
the totally enclosed FDA approved automatic system (Sepax,BioSafe America, Inc.). The final
product, re-suspended with cell culture medium (DMEM+10% albumin and 10% autologous plasma)
in a volume of 200 mL, will be placed in a 600 mL transfer bag. 2 mL of the product will be
taken from the bag before clinical use to test sterility using culture media and
immunofluorescent cytometry to characterize cell type and viability.
Two days before the transplant, the patient will begin "boosting" therapy to mobilize cells
by means of systemic injections of analogous recombinants of granulocyte colony-stimulating
factor (GCSF)(Granocyte, 1 M IU/kg (max. 15 M IU) and Erythropoietin, 400 IU/kg
(max.6,000IU). These will be injected for the two days prior to surgery.
InBreath Bioreactor (the special bioreactor for cultivating trachea) The work in the current
protocol will involve a bioreactor design previously utilized by Macchiarini P. ang colleges
in a successful first-in-man implantation of a tissue-engineered large airway replacement.
The device, commercialized under the name, InBreath 3D Organ Bioreactor (Harvard Bioscience,
Inc.) is designed for placement within a tissue culture incubator and consists of a modular
polysulphone organ chamber, motor unit and remote controller. The chamber is easily
detachable from the motor unit and its polysulphone construction permits sterilization with
the standard gas plasma sterilization process that is readily available in the operating
room in Peoria. The motor unit provides consistent rotation to the tissue holder within the
chamber, ensuring controlled application of hydrodynamic shear forces to the developing
tracheal construct. A fully enclosed motor housing protects the brushless motor from the
corrosive moisture within the incubator. The remote control unit is placed outside the
incubator providing a means to adjust rotational speed without disturbing the incubator
environment.
The seeded construct was allowed to incubate in the bioreactor for 96 hours prior to removal
for implantation. Based on the five previous adult cases using the POSS-PCU (Polyhedral
oligomeric silsesquioxane-poly(carbonate-urea) urethane), PET(Polyethylene terephthalate)
and PET:PU (Polyurethane) synthetic scaffolds, the internal and external surfaces of the
scaffold will be seeded with the freshly isolated bone marrow mononuclear cell fraction. The
bioreactor will be started with an initial speed of 0.5 cycles/min for 18 hours (then
stepwise increase up to 2.5 cycles/min). Incubation will be during the 48 hours preceding
the transplant procedure. This incubation protocol worked very well in the previous cases
using the three different synthetic nanocomposite tracheal scaffolds.
Cultivation steps:
The tracheal reseeding procedure will be done in our aseptic culture GMP (Good Manufacturing
Practice) facility that was established and fully functional.
1. Isolated MNC will be prepared according to the Sepax 2 protocol for bone marrow
separation and resuspended in a 300ml bag containing 0.9%Normal saline solution (with
10% human albumin).
2. The sterilized scaffold (gamma irradiation sterilization), the bioreactor (plasma
sterilization) and surgical instruments (autoclaved) will be placed into the laminar
hood.
3. All persons that are manipulating the cells/bioreactor and scaffold will be fully
trained in have GMP grade standards, namely sterile gloves, specific overalls, etc.
4. The bioreactor will be opened inside the hood in sterile conditions and placed on a
sterile tissue. The scaffold will be mounted on the organ holding fixtures and placed
into the bioreactor. Once the scaffold is transferred and fixed into the bioreactor the
MNC (+DMEM plus albumin and autologous plasma) will be seeded on the scaffold´s
surface. Medium (including autologous plasma and human albumin) will be added to the
bioreactor chamber to a total volume of 200 ml.
5. The factors will add to medium: 39.3 ng/mL (100 nmol/L) dexamethasone, and 10 μg/mL
insulin.
6. Then the bioreactor chamber (including the scaffold, MNC + 200 ml of medium) will be
placed into the incubator and mounted onto the motor unit of the bioreactor (previously
placed inside the incubator).
7. The bioreactor will be started with an initial speed of 0.5 cycle/min for 18h (then
stepwise increase up to 2.5 cycles/min).
8. After 24h, an additional 50ml of the prementioned medium will be added to a total
volume of 250ml inside the chamber. At this time a small aliquot of chamber fluid will
be tested with gram stain and injected into culture media to check for contamination.
9. After 48 hours the chamber will be opened and an aliquot will harvested for culture and
Gram stain. A small biopsy of the neotrachea will be taken for the MTT viability test.
Once it is determined that the cells are viable and there is no sign of media
contamination (Gram stain and interim reading of direct inoculation culture) the
trachea will be deemed ready for implantation and the patient will be placed under
anesthesia and the surgical procedure will be started.
Day of transplantation:
Intra-operative Surgical Procedure The morning of the transplant the graft will be tested
for cell growth (MTT test and for sterility by gram stain and analysis of interim culture
results). Once the graft is deemed ready for implantation, the patient will be placed under
general endotracheal anesthesia.
Thoracic and abdominal procedures Having performed the resection of the airway's damaged
segment, the airway construct will be seeded intraoperatively with the respiratory cell
biopsies on the internal surface. The graft will be then injected (conditioned) with growth
factors including 10 ng/mL of recombinant human transforming growth factor-β 3, 10 nmol/L
recombinant parathyroid hormone-related peptide, 100 nmol/L dexamethasone, and 10 µg/mL
insulin, GCSF (10 µg/kg) and Erythropoietin (40,000 UI) (to stimulate the mobilization of
the peripheral hematopoietic cells). The implant will be then anastomosed proximally and
distally so as to reconstruct the airway defect using sutures. It will be then covered and
wrapped by an omentum major flap (adipose vascularized tissue detached from the large bend
of the stomach, harvested on the right or left gastroepiploic artery and then carried over
to the mediastinum trans-diaphragmatically or sub-sternally), to guarantee long-term
protection of the graft and of the anastomosis and obtain indirect graft's
neovascularisation.
After transplantation:
Post-operative treatment
To boost the regenerative process, the patient (current weight about 13 Kg) will be treated
pharmacologically in the post-op period by systemic injections of:
1. Analogous recombinants of GCSF (Granocyte, 10 million IU/kg up to a maximum of 30
million IU)
2. Analogous synthetics of Erythropoietin (Epoetin alpha or beta 40,000 IU)
Both factors will be administered in suitable concentrations to stimulate the
mobilization/recruitment of hematopoietic cells, in "regenerative" doses which have not been
associated with any side-effects. Every second day the plasma Erythropoietin level and the
blood count (including haemoglobin and white blood cell counts) will be monitored.
Haemoglobin levels greater than 15 g/dl will raise concerns for hyper-viscosity and prompt
removal of 10-20 cc/kg of blood and may prompt the addition of a continuous infusion of
heparin to keep the Activated Partial Thromboplastin Time (APTT) levels between 40-60
seconds. White blood cell levels above 50-60,000/μl will be considered "toxic" and will
result in a reduction/suspension of the GCSF therapy until numbers fall below 30,000.
Treatment with GCSF and Erythropoietin will be carried out every other day for 2 weeks
following the transplant according to the following table:
Follow-up
The follow-up will be carried out at the Cardiothoracic Surgery Department of the Krasnodar
Regional Hospital, and will include:
1. Endoscopic evaluation (flexible and/or rigid bronchoscopy) of the transplanted airway
every day for the first week and every other day for the second week, after which once
a month for the first six months, and every 6 months thereafter for the first 5 years.
2. Evaluation of the blood count with white blood cell formula daily for the first two
weeks.
3. Evaluation of mobilized progenitor cells from peripheral blood every second day during
2 weeks.
4. Immunogenic evaluation. After 3, 7 and 30 days from the transplant, a blood sample will
be taken to make a study of the histocompatibility by evaluating the antibodies. The
immunogenic follow-up will also be carried out after 3, 6 and 12 months from the
transplant.
5. Post-operative Tobramycin inhalation (2x5ml/day for 30 days) to prevent from pneumonia
and graft bacterial contamination.
6. Computerized tomography of the neck and chest with a three-dimensional reconstruction
of the transplanted airway will be done at month 1, month 3 and month 6 of the
follow-up, and every 6 months thereafter for the first 5 years.
