**7.3.5 Intraarticular injection**

On postoperative day 7, 8 mL of the fresh PBPCs is mixed with 2 mL of HA and injected into the operated knee joint under aseptic conditions in the out-patient clinic. Prior to this, the knee is first aspirated for hemarthrosis. At weekly intervals, 8 mL (from two 4 mL cryovials) of the frozen PBPC were obtained from the laboratory, allowed to thaw to room temperature, mixed with 2 mL of HA, and injected into the operated knee joint for 4 subsequent weeks. A flow chart of the protocol for patients undergoing articular cartilage regeneration is shown in Fig 14. Table 2 shows the PBPC count of 20 recent consecutive

Fig. 12. A typical apheresis process.

**7.3.5 Intraarticular injection** 

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

On postoperative day 7, 8 mL of the fresh PBPCs is mixed with 2 mL of HA and injected into the operated knee joint under aseptic conditions in the out-patient clinic. Prior to this, the knee is first aspirated for hemarthrosis. At weekly intervals, 8 mL (from two 4 mL cryovials) of the frozen PBPC were obtained from the laboratory, allowed to thaw to room temperature, mixed with 2 mL of HA, and injected into the operated knee joint for 4 subsequent weeks. A flow chart of the protocol for patients undergoing articular cartilage regeneration is shown in Fig 14. Table 2 shows the PBPC count of 20 recent consecutive 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.


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+ counts, and cell viability.

Articular Cartilage Regeneration with Stem Cells 147

Five cases are presented here with their respective chondral biopsies and histology. These cases provided explanation to the principles of chondrogenesis in our novel approach. The patients are part of a larger pilot study in which 180 patients who presented with chondral defects of the knee joint were recruited. Postoperatively, the clinical course of these 5 patients presented an opportunity for a second-look arthroscopy. Two patients underwent contralateral knee surgery, and one patient had removal of a Tomofix plate and screw construct (Synthes, West Chester, PA), providing an opportune setting of anesthesia for second-look arthroscopy. One patient had recurrence of discomfort attributed to a prominent osteophyte and elected for a further arthroscopic procedure. The last patient had returned to football 18 months after articular cartilage repair and sustained a torn anterior cruciate ligament of the previously treated knee. He elected for arthroscopic reconstruction, which provided an opportunity for second-look arthroscopy. Informed consent after discussion of risks and benefits, as well as local ethics committee approval, was obtained

During the second-look procedures, a chondral core biopsy specimen was procured. This was performed arthroscopically with a 5.5 mm sterilized BioCorkscrew anchor driver (Arthrex, Naples, FL). Typically, a 2 mm diameter specimen of cartilage together with a core

Arthroscopically, the regenerated articular cartilage appeared smooth and had excellent integration with the surrounding native cartilage without any delamination or hypertrophy. The exception was case 2, in which the drill holes over the lateral patellofemoral joint were too far apart with resultant tufts of cartilage seen between areas devoid of regenerated

Fig. 15. Solid articular cartilage core biopsy with a 2 mm diameter including the underlying

**7.4 Introduction to clinical cases** 

**7.4.1 Second-look arthroscopy with chondral core biopsy** 

of bone up to 1 cm in length is obtained (Fig 15).

before biopsy.

cartilage (Fig 16).

subchondral bone.

Fig. 14. Flow chart showing the standard protocol for articular cartilage regeneration with PBPC and HA.

Fresh cells are used preferably for the first injection because of a mean viability of 99% compared with frozen cells, which have a mean viability of 87%. It should be noted that 8 mL of PBPC injected into the operated knee has a mean of 20 million CD105+ cells. Historically, the cell marker CD34+ (hematopoietic stem cells) has been used to identify functional cells for bone marrow transplant. We have begun to draw interest in CD105+ cells, because this is the marker for mesenchymal stem cells.

Five weekly injections are based on the HA protocol for osteoarthritis, as well as the suggestion from preclinical animal studies involving Bone Marrow Progenitor Cells (BMPC) that an increased number of intraarticular injections is more efficacious (Saw et al, 2009). Table 3 shows the viability of 5 consecutive frozen PBPC samples after mixing with HA. As can be seen, there is no effects of HA on the viability of the PBPC.


Table 3. Cell count viability.
