**7.3.3 Postoperative rehabilitation**

142 Modern Arthroscopy

infected knees, gross bone defects, rheumatoid arthritis, or intraarticular corticosteroids within the previous 6 weeks; and gross valgus or varus deformity not correctable during surgery.

All surgical procedures were performed by a single surgeon (by the first author) involving standard arthroscopic techniques in the supine position without a tourniquet. Saline solution irrigation bags were chilled in an ice-water bath before use to minimize bleeding during the arthroscopic procedure. In our experience, we have had difficulty performing microfracture to the patella and areas of the plateau. For this reason, our preferred method is arthroscopic subchondral drilling modified from the principles established by Steadman et al (Steadman et al, 1999), for microfracture and Pridie (Pridie, 1959) for drilling. We begin by defining the extent of cartilage injury with a probe. A 3.5 mm full-radius shaver is used to debride loose cartilage to a stable margin; often a straight or curved arthroscopic biter is required as well. A 2 mm burr, with its guard removed, "drills" from the surface of the defect to the bone marrow, creating a conduit. The remaining area within the margin is also drilled to a depth of 5 to 10 mm. Initially, we spaced drill holes 3 to 4 mm apart. The methods have subsequently been refined such that a goal of 1 to 2 mm between drill holes is now preferred based on the results of second-look arthroscopy. It is not crucial that the subchondral drilling be performed perpendicular to the bone surface because a lesser angle of drilling capable of penetrating into the subchondral bone is sufficient. Abrasion chondroplasty up to a depth of 1 mm is performed with burring of the bony area between drill holes. The result is an extended area of bleeding bone, hence a larger surface area for the initiation of articular cartilage repair with PBPC and HA (Fig 10). The arthroscopic portals are closed with No. 3-0 nylon suture. A mixture of 20 mL of 0.5% bupivacaine hydrochloride and epinephrine, 3 mL of 1 mg/mL morphine, and 2 mL of HA (Hyalgan; Fidia Farmaceutici, Abano Terme, Italy) is injected into

Fig. 10. Subchondral drilling. (A) A delaminated flap tear on the medial & central trochlear of a left knee. (B) A 2 mm burr with guard removed allows for drilling with light suction.

(C) View after debridement, subchondral drilling and abrasion chondroplasty.

**7.3.2 Surgical procedure** 

the operated knee at the end of the surgical procedure.

Cold therapy is initiated for one hour 2 to 3 times a day immediately in the postanesthesia period and continued throughout the first month after surgery. On the first postoperative day, continuous passive motion is used on the operated knee for a duration of 2 hours (Fig 11). This is continued daily for a period of 4 weeks. The range of motion is initially set from 0° to 30° and progresses as the clinical situation improves. Patients with subchondral drilling to the weight-bearing femorotibial joint are instructed on crutch-assisted partial weight-bearing (15 to 20 kg) for the first 4 weeks. This progresses to full weight-bearing in 6 to 8 weeks. Patients with drilling to the patellofemoral joint are allowed full weight-bearing as tolerated with restrictions from weight-bearing on stairs for the first 3 months after surgery. This is to avoid overloading the patellofemoral joint.

Fig. 11. Patients undergoing postoperative rehabilitation (A). A closer view of patients showing continuous passive motion applied on the operated knee (B).

## **7.3.4 Neupogen administration, apheresis, and cryopreservation**

Human granulocyte colony–stimulating factor is a glycoprotein that regulates the production and release of functional neutrophils from the bone marrow. Neupogen contains recombinant granulocyte colony–stimulating factor and causes marked increases in peripheral blood neutrophil counts with a minor increase in monocytes within 24 hours. On postoperative days 4, 5, and 6, patients were given a morning dose of 300 micro-grams of Neupogen (Filgrastim, Amgen, Thousand Oaks, CA) subcutaneously. On postoperative day 7, autologous PBPC were collected by an automated cell separator (apheresis) via central venous access. Venous access was achieved through a femoral double-lumen catheter placed into the contralateral leg, under ultrasound guidance, performed by a trained specialist. Apheresis was performed by use of the Spectra Optia Apheresis Machine (Caridian BCT, Denver, CO). A fresh aliquot of 8 mL of PBPC was separated for fresh intraarticular injection into the operated knee. The remaining PBPC were cryopreserved in 10% dimethyl sulfoxide and divided into 4 mL cryovials for storage in liquid nitrogen at –196°C. Flow cytometry with CD34+ (hematopoietic stem cells) and CD105+ (markers for mesenchymal stem cells) was quantified. Flow cytometry was performed with a Beckman Coulter FC500 device (Beckman Coulter, Fullerton, CA). Fig 12 showing the apheresis process. The cryopreservation of the harvested PBPC in cryovials can be seen in Fig 13.

Articular Cartilage Regeneration with Stem Cells 145

patients after refinement of our processing methods, showing data of fresh and frozen

samples with white blood cell count, CD34+ and CD105+ counts, and cell viability.

CD 34+ (106) Cells per 4ml Vial

AVERAGE 32.06 2.38 1.57 9.28 10.76 99.12 87.29

Table 2. Example of 20 consecutive PBPC counts from patients following 3 neupogen injections. Data of fresh and frozen samples with white cell count, CD34+ and CD105+

CD 105+ (106) Cells

Fresh Frozen Fresh Frozen Fresh Frozen

32.00 6.86 4.58 7.42 8.14 99.30 79.90 24.40 1.04 0.66 9.98 14.41 99.30 89.65 30.00 0.33 0.23 3.32 5.08 99.10 89.99 35.60 1.03 0.74 15.44 20.33 99.01 91.97 14.80 3.88 2.86 2.69 8.87 99.40 87.90 33.50 0.44 0.37 8.95 7.04 99.60 95.30 33.50 3.28 2.33 5.41 7.75 99.00 82.90 34.80 2.02 1.34 13.60 14.01 99.50 94.04 23.00 2.53 1.69 7.01 13.77 99.50 88.10 37.10 2.42 1.32 3.37 5.72 98.90 88.10 26.50 1.55 1.07 4.54 6.98 99.00 84.80 52.10 2.76 2.17 8.89 4.56 99.40 90.30 38.80 3.15 1.82 15.50 11.62 99.30 79.30 32.10 0.86 0.75 12.79 6.35 98.90 91.30 33.00 5.71 3.14 6.30 8.53 99.10 78.40 20.00 1.02 0.62 14.80 15.40 97.80 88.40 37.50 3.19 2.08 4.15 1.66 99.30 82.20 26.90 2.78 1.90 1.43 4.44 99.30 82.30 31.00 1.55 1.01 23.45 31.15 99.10 89.10 44.60 1.16 0.67 16.62 19.40 98.50 91.80

per 4ml Vial Viability %

White Cell Count 10³ / uL

counts, and cell viability.

Fig. 12. A typical apheresis process.

Fig. 13. Cryopreservation of the harvested PBPC in cryovials.
