Mesenchymal stem cell (MSC) is one kind of stem cell which is gained form adult tissue. Although MSC derived from autogenic bone marrow are proven to help regeneration in non union fracture and long bone defect, the aspiration process through iliac crest is invasive and painful.

Therefore, alternative source of MSC which is less invasive is needed. Adipose and umbilical cord is a "waste product" that proven to contain enormous MSC. Furthermore adipose and umbilical cord as an allogenic source is more abundant in number compares to autogenic bone marrow. This enormous source need and adequate preservation technique before applied to the patient. According to that, researchers want to explore the potency of MSC from bone marrow, umbilical cord and adipose as the source of allogenic MSC and the effect of cryopreservation technique to the viability and quality of MSC. We will also compare the effectivity of MSC implantation from bone marrow, umbilical cord and adipose applied to non union fracture and long bone defect.

Samples from bone marrow, umbilical cord and adipose are cultured and the viability of the cells are observed. Some of the cells are implanted directly to the patient with non union fractures and long bone defect while some are cryopreserved in liquid nitrogen -190 degree Celsius in three months. All samples are thawed and the viability of the cells are observed. Patient who are implanted by MSC allogenic will undergo clinical and radiological examination in the third, sixth and twenty second month after implantation.

Research Methods Study Design This study is an experimental one arm study post test only. Estimated Study Time Research estimated time would be 24 months, from May 2014 to May 2016. Sample Gaining Procedure This research is a pilot study. Samples are obtained consecutively from all source population that meet the criteria. The number of samples from each mesenchymal stem cell ( adipose, bone marrow and umbilical cord) source are three. Each samples derived from three different donors that met the inclusion criteria. For the implanation, the subjects are five from each intervention. Yet regarding the limited funding source and time, we will recruit one subject for each kind of MSC.

Subject Criteria Inclusion criteria for MSC donor

Bone marrow donor :

Male/female aged 19-30 year without any comorbiditites (Diabetes mellitus, cardiovascular and any other autoimmune disease), HIV test Hepatitis B test and Hepatitis C test are negaitve, no fungal and bacterial contamination in the bone marrow. Subjects are willing to be aspiratied in the iliac crest in order to get the bone marrow.

Adipose donor :

Adipose tissue are gained from liposuction or open reduction internal fixation procedure. Samples of adipose are free from HIV, Hepatitis B, Hepatitis C and free from fungal and bacterial contamination.

Umbilical cord donor :

Umbilical cord are form elective seccio caecaria from a fullterm mother without any complications and free from HIV, hepatitis B, hepatitis C and no fungal and bacterial contamination.

Recipient inclusion criteria Critical bone defect patients aged 6-55 who are willing to undergo surgical intervention.

Recipient exclusion criteria Patients with pathological fracture caused by malignancy, immunocompromised ( HIV AIDS, Diabetes mellitus, active Hepatitis), in a immunosuppresant therapy ( chemotherapy or steroids).

Drop out criteria Patients are ruled out from this study if he/she stated to do so in the time this research are held or she/he undergoes any other threatment that are not related to this study. Patient who does not show any clinical improvement in three consecutive months is categorized as failed to threat. All drop out and failed to threat patient could get other threatment.

Informed Consent All subjects must fill and sign in the informed consent letters.

Research Protocol Mesenchymal stem cell taking method Bone Marrow taking Patient is lying down in supine position, anesthetized locally. Aseptic and antiseptic are done in the illiac crest location. Aspiration needle is inserted 450 to horizon in illiac crest. Hub is released and a 10 cc syringe that contain heparin is connected to te needle. We aspirated about 50 cc bone marrow from each subject.

Umbilical cord taking Right after the delivering the baby, the umbilical cord are cut and kept in a sterile bowl containing 0.9% NaCl in 40 until the sample is proceed.

Adipose taking Adipose tissue are derived from liposuction or open reduction internal fixation procedure. It is kept in a sterile bowl containing NaCL 0.9% in 40 C. Processing of the sample is done within 8 hours after sample are taken.

Cryopreservation and re activation. All the samples are taken to culture laboratory in integrated service of stem cell medical technology Cipto mangunkusumo hospita.this laboratory is GMP (good manufacturing Product) certified. The samples ate cultured in appropriated medium until it reach confluence and harvested. The cells then undergo caracterisation test by flow cyto meter and viability and numbers are counted. Some of the cells then cryopreserved while some are directly implanted into patient. The cells are cryopreserved for three months and then reactivated. Viability and numbers then are measured. The cells then are implanted to non union patient.

Specimen sterility Sterility tests are done three times to ensure there is no fungal and bacterial contamination.

HA-CaSO4 and MSC For every centimeter of defect, 10 millions cells and 50 pellet HA-CaSO4 are needed. The diluted MSC then mix with the HA-CaSO4 and incubated 5 minutes before implanted.

Intervention Surgical intervention is needed to assemble the fixation device in the long bone. During the surgery, pellet HA-CaSO4 are inserted into the defect. After soft tissue are closed the rest of the serum is injected into the defect area.

Observation and follow up Clinical and radiological follow up is done every 4 weeks. Observation is done for 12 months or untill the bone unites. Every subject will be followed up in the third, sixth, twelfth and twenty forth or until the fixation device is taken off.

Recipient criteria for non union fracture

Inclusion criteria for MSC donor

• Bone marrow donor : Male/female aged 19-30 year without any comorbiditites (Diabetes mellitus, cardiovascular and any other autoimmune disease),HIV test Hepatitis B test and Hepatitis C test are negaitve, no fungal and bacterial contamination in the bone marrow. Subjects are willing to be aspiratied in the iliac crest in order to get the bone marrow.
• Adipose donor : Adipose tissue are gained from liposuction or open reduction internal fixation procedure. Samples of adipose are free from HIV, Hepatitis B, Hepatitis C and free from fungal and bacterial contamination.
• Umbilical cord donor : Umbilical cord are form elective seccio caecaria from a fullterm mother without any complications and free from HIV, hepatitis B, hepatitis C and no fungal and bacterial contamination.

exclusion / Drop out criteria

-Patients are ruled out from this study if he/she stated to do so in the time this research are held or she/he undergoes any other threatment that are not related to this study. Patient who does not show any clinical improvement in three consecutive months is categorized as failed to threat. All drop out and failed to threat patient could get other threatment.

inclusion criteria for recipient : -male/female aged 6-55 year old with bone critical defect

exclusion criteria for recipient :

-Patients with pathological fracture caused by malignancy, immunocompromised ( HIV AIDS, Diabetes mellitus, active Hepatitis), in a immunosuppresant therapy ( chemotherapy or steroids).