TISSUE ENGINEERING IN ORTHOPAEDICS

 

DR S K SHARMA

ASSOCIATE PROF.ORTHOPAEDICS

HEAD OF DEPTT.OF ORTHOPAEDIC SURGERY

R.D.GARDI MEDICAL COLLEGE , UJJAIN MP INDIA

 

ADDRESS:-

O r t h o n i c

Opp. Narendra Cinema

UJJAI N MP 456001

Tel:0734 2553832 and 2519019.

Mobile :94251 95613

 

e-mail:orthonik@yahoo.com

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TISSUE ENGINEERING IN ORTHOPAEDICS

The healing of fractures in bone is the most common happening where bone regenerates due to its unique ability . Nonetheless healing without problem ,restores the rapid rehabilitation.  A few goes to non-union and requires autologous bone marrow transplantation to enhance the healing. On the other hand, bone marrow and osteonecrosis of  head of  femur is a complex phenomenon. MRI scans of proximal femur showed  bone marrow edema in as early as 12 weeks  after the initial trauma to hip .This is bone marrow edema syndrome ,characteristic of  early diagnosis of AVN of femur. There is reversal of amount of osteogenic cells to adipocytes in the patients of osteonecrosis ;that is why the yellow marrow becomes in abundance. Steroids also stimulates adipogenesis. Adipocytes and osteoblasts share a common pool of stem cells. Under  the effect of steroids , pluripotent stem cells produces the insufficient number of osteoblasts to meet the needs of bone remodeling ; ultimately leads to AVN. . In 1869,Goujon , first demonstrated the osteogenic capacity of bone marrow in rabbits.

 The presence of isolated injuries to the cartilage of the knee is said to be extensive for osteoarthrosis because the repairing is limited due to the nature of chondrocytes. It is  avascular and aneuronal structure.Full thickness defects of moderate to large [1.3 to 12.0 square cms.] fail to heal , conservatively. The persistence of untreated focal chondral lesions is primarily caused by the lack of  chondrioprogenitor cells within the defect. The use of autologous cultured chondrocyte transplantation was initiated in 1980s.Its clinical use was pioneered  in Sweden by Lars Peterson in 1987.

 

AUTOLOGOUS BONE MARROW TRANSPLANTATION

The adult skeleton possesses two types of bone marrow, red and yellow. The red marrow is haematopoietically active and still persists in the iliac crests. The haematopoietic stem cells in bone marrow has been extensively used clinically in cases of non union and osteonecrosis of bones; like AVN of head of femur, due to its therapeutic relevance in transplantation. These cells are pluripotent and capable of  producing progeny that can  differentiate into any and all of the cells of circulating blood and the immune system  i.e.Autogenous Connective Tissue Progenitors (ACTP) ;through a well defined series of steps. This red marrow also contains a stroma of osteogenic precursor cells ; lebelled as tissue progenitor , more specifically osteogenic progenitors. That is why bone marrow is potential to lead to effective bone regeneration after implantation .In 1869, Goujon , first demonstrated the osteogenic capacity of bone marrow in rabbits. In clinical practice autologous marrow is harvested from iliac crest and immediately transplanted to the site in need of skeletal repair . This can be done in OPD also .

 

ACTP

The technique of transplantation of  ACTP is the first of four major cell based strategies of future tissue engineering. The ACTP may be staged to one of the following four :-

1.transplantation of ACTP

2.Targetting local connective tissue progenitor with growth factors where new tissue is required.

3.Transplantation of culture-expandedd or modified ACTP

4.Transplanting fully formed tissue generated in vitro.

 

 

 

TECHNIQUE OF MARROW ASPIRATION

From lateral approach a 6-8 cms long and 1.5mm in internal diameter a bevelled needle is inserted deep into the spongy iliac bone . All aspirate is collected in plastic bag via 10cc plastic syringe. Plastic bag contains cell culture medium and anticoagulant solution[citric acid,sodium citrate,dextrose]. The aspirated maroow is even reacher in stem cells when it is aspirated in small fractions , which reduces the degree of dilution by peripheral blood .

The bone marrow aspirate filtered to separate the cellular aggregates and fat. Then the concentration of aspirate is used to inject at the site. Two techniques are there to inject ; one is aspiration,concentration and re-injection during the same operation ; within one hour. Other one is freezing technique, which allows the marrow to be re-injected at a later date; with no time limit. The early use of aspirate is concentrated in a cell separator at a 5-minute centrifuge.

 

TECHNIQUE OF INTRA-OSSEOUS  INJECTION OF BONE MARROW

Bone marrow aspiration needle with trocar is used to place the aspirate at the site of pseudoarthrosis, fracture ends. A small Mazabraud trephine is used for implantation in osteonecrotic segment. The tip of trocar is positioned by using a brightness amplifier.An opaque medium Hexabrix injected to check the bone marrow has been introduced in the bone.The marrow aspirate is injected slowly at a rate of 20 ml.per minute.When injection is finished the trocar is gradually withdrawn.

 

RESULTS

P.Hernigou, 2005;July ,has clinical experience with aspiration of bone marrow in more than 1000 patients of non-union and osteonecrosis. No complications were encountered except theoretical criticism of fat embolism found in study on dogs. Josefson described first  successful  intra-osseous injections of therapeutic substance , in 1934 for pernicious anaemia. Shock in children was treated by physiological serum injected into bone-marrow , in 1940 by Tocantins. The influence of the number of progenitors on the results of treatment of AVN Hip and NU  fracture tibia . The number of  fibroblastic colony –forming units (CFU-Fs) in bone marrow was determined by assessing the number of nucleated cells and the prevalence of CFU-Fs among them.

 

TECHNIQUE  OF AUTOLOGOUS CHONDROCYTE IMPLANTATION [A.C.I.]

Initially during arthroscopy defect was examined and slivers of cartilage[300-500mg] were obtained in a sterile glass tube containing 0.9% NaCl ; from the upper minor load bearing area of the medial femoral condyle in the injured knee for culture to the cell-culture laboratory and later implantation. Blood was collected from the patient preoperatively for serum used in culture. The cartilage pieces were minced and washed in Ham’s F-12 medium in culture bottle. The isolated cells were resuspended in Dulbecco’s modified Eagle medium/F12/1:1 with the addition of 10 % of patient’s own serum. The cells were incubated in 25-sqcm tissue-culture flask in 7% CO2 in air at 37ْ C. After one week the cells were suspended by means of trypsin-EDTA and transferred to wash with Ham’s medium. The cells were released for implantation if the cell viability is more than 85% as determined by trypan blue staining. The cells ,now being taken into the tuberculin syringe ; ready for arthroscopic implantation in the patient’s knee.

Under general or spinal anaesthesia and tourniquet; medial or lateral parapatellar arthrotomy was performed. The chondral lesion debrided back to the best cartilage  available .The periosteal flap harvested from the proximal medial subcutaneous tibia. The flap fitted and sutured to the surrounding rim of debrided cartilage with interrupted 5-0 or 6-0 Vicryl. The rim sealed with fibrin glue except for one corner , where the cultured prefilled autologous chondrocytes were injected into the defect. After the cell injection ,this open corner was sutured and sealed.Closed and covered by elastic bandage. Continuous passive motion administered for 48 hrs, postoperative . On crutches 8 weeks ,progressing to full weight bearing by 10-12 weeks.

 

EVALUATION

The clinical assessment was done on knee scoring systems described by Lysholm and Gillquist and others. The mechanical characteristics of  repaired tissue were assessed by Electromechanical indention probe. The histological evaluation done by  a 2-mm core biopsy instrument.

 

RESULTS

In the series of Peterson , Sweden the long term durability of ACI was reported to be 85% for 2 years. Patients returned to normal activities of daily living and sport by 2 years after ACI.  Sjogren , 2005 ;used ACI with the periosteal combination;use of homogenous clone on agarose  produced good to excellent articular repair.  Articulr chondrocytes are able to form clones of different properties in agarose and the periosteum has a capacity of stimulating chondrocytes clonal growth  which secrets significant amount of IL-6,IL-8,GM-csf and TGF-beta .

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 REFERENCES:-

 

 

1. Goujon,E. Abstract .J L Anat.1869;6:399

 2. Garg NK ,Gaur S , Sharma, S. Percutaneous autogenous bone marrow grafting in20 cases of un-united fractures . Acta Orthop Scand 1993;64:671-2.

3. Hernigou P,Beaugean, F. Pseudoarthrosis is treated by percutaneousautologous bone marrow graft.Rev Chir Repartrice Appar Mot 1997;83:495-504.

4. Brittberg M, Sjogren-Janssen E, Peterson , L : Clonal growth of human articular cartilage and the functional role of the periosteum in chondrogenesis.Osteoarthritis cartilage. 2005 Feb.;13(2):146-53.

5.Peterson Lars , Mats Brittberg , Illka Kiviranta, ACI –Biomechanics and long term durability. ClinOrthop. 374:212-234. 2000 [medline]

6. Goel, S C . Editorial : Stem cells in Orthopaedics . IJO; April 2005;39-[2],73-74.